Protein and protein conjugate for diabetes treatment, and applications thereof

ABSTRACT

The present invention relates to the field of biopharmaceuticals, and in particular to a protein, a protein conjugate, a pharmaceutical composition and its use for treating diabetes. The fusion protein of the present invention is obtained by linking two polypeptides, wherein one polypeptide is an interleukin-1 receptor antagonistic protein or an analogue thereof, and another polypeptide is GLP-1 receptor binding polypeptide or an analogue thereof, or an insulin receptor binding polypeptide or an analogue thereof, or a GIP receptor binding polypeptide or an analogue thereof. The fusion proteins of the present invention and conjugates thereof have a significant efficacy in treating diabetes, and can be used in a lower dose, resulting in marked reduction in side effects.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser. No. 15/585,655 filed May 3, 2017, which is a divisional of U.S patent application Ser. No. 14/402,074 filed Nov. 18, 2014, now U.S. Pat. No. 9,745,359, which is a national phase entry under 35 U.S.C. § 371 of International Application No. PCT/CN2013/000587 filed May 17, 2013, published in Chinese as International Publication No. WO 2013/170636 A1, which claims priority from Chinese patent application 201210157196.6 filed May 18, 2012, all of which are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 31, 2015, is named 9BOSS3.3F-002(E)_SL.txt and is 1,095,257 bytes in size.

TECHNICAL FIELD

The invention belongs to the field of biopharmaceutics, and relates to a protein, a protein conjugate and the use thereof for treating diabetes.

BACKGROUND ART

Cytokines are biologically active macromolecules present in or released from cells. Most of them are polypeptide-like species, such as interleukins, interferons, colony-stimulating factor (CSF), and tumor necrosis factor (TNF). They are not expressed or expressed in a low amount in normal body, but are transcribed and translated in abnormal states (disease or infection). Cytokines regulate or modulate cellular responses in inflammatory or immune responses. They activate biological responses by binding to the specific receptors on the surfaces of the sensitive cells. Since cytokines often have their receptors on various target cells, pleiotropic effects of the responses are common features for a cytokine. A second messenger can also be generated through other cytokines or cytokine receptors.

For a highly active cytokine, there must be a mechanism by which its release is controlled and its activities are constrained. The biosynthesis and release of a cytokine can be regulated strictly by other cytokines or exogenous factors. Two mechanisms for controlling the activities of a cytokine have been found: the first is a receptor antagonist (inhibitory protein type I) which has a structure homologous to that of the cytokine and can bind to the receptor molecule, but does not trigger signal transduction, thus exerting inhibition by competing with the cytokine; the second is a soluble receptor molecule (inhibitory protein type II) which competes with the cellular receptor for cytokine binding, thus exerting inhibition. A large amount of experiments have shown that the two suppressive responses have a physiological correlation. At present, it has been believed that inhibitor type II has a neutralizing effect similar to that of a buffer solution, which in turn limits the systemic effect of a cytokine. Meanwhile, it also allows the cytokine to rise locally to a high concentration, thereby enhancing a paracrine effect. Their recombinant forms have pharmacological activities.

Interleukin 1 (IL-1) is involved in a wide variety of physiological processes. By inducing secretion of the neutral proteases and other cytokines (tumor necrosis factor TNF), IL-1 stimulates proliferation of various hematopoietic cells and other cells, regulates pro-inflammatory responses, and mediates damage to tissues in an inflammation, including stimulating proliferation of the synoviocytes and the chondrocytes and production of PGE2, collagenase, phospholipase A, etc, inducing inflammation in a joint; facilitates release of neutrophils from the bone marrow, induces chemotaxis and infiltration of mononuclear cells and the multinucleated granulocytes into an inflammatory site, releases lysosomal enzymes locally, causes de-granulation of the basophils and the mast cells, releases inflammatory mediators, etc. IL-1 can impose damage directly on β cells which produce insulin in the pancreatic islet. The diseases associated with IL-1 comprise rheumatoid arthritis, diabetes mellitus, systemic lupus erythematosus, dermatosclerosis and other immune diseases.

IL-1 includes two relevant factors: IL-1α and IL-1β. Furthermore, there is a relevant polypeptide known as IL-1 receptor antagonist (IL-1Rα). Both IL-1α and IL-1β exert their biological activities by binding to IL-1 receptor (IL-1R) on the cellular surface and signaling into the interior of a cell via a signal transduction system. Two types of IL-1R have been found: type I receptor (IL-1RI) and type II receptor (IL-1R II).The type I receptor, also known as T-cell receptor, has a signal transduction function; whereas the type II receptor, also known as B-cell receptor, can bind to IL-1, but does not transduce the signal. In fact, IL-1RII acts as an attenuator of IL-1, which can be designated as a “trapping” receptor. When IL-1 binds to IL-1R I, a complex is formed and then binds to a IL-1R accessory protein (IL-1R AcP) with a high affinity. The IL-1 signal transduction is activated probably by a heterodimer formed by association of the intracellular portion of the IL-1R I with the IL-1R-AcP. Furthermore, the extracellular portions of the IL-1R are also known as the soluble receptors, that is, type I soluble receptor (sIL-1RI) and type II soluble receptor (sIL-1RII), which exist in the circulation system of the body both in the normal state and in the diseased cases and can bind to IL-1α, IL-1β, or IL-1Rα to act as a natural “buffer”.

Arend, et al. (“Interleukin 1 receptor antagonist: A new member of the interleukin 1 family.” J Clin Invest, 1991,88(5): 1445-1451) has found a substance present in the cell culture supernatant and the body fluid and having an activity of inhibiting IL-1 and designated it as IL-1Rα. Eisenberg, et al. (“Interleukin 1 receptor antagonist is a member of the interleukin 1 gene family: evolution of a cytokine control mechanism.” PNAS, 1991, 88(12): 5 232-5236) has found by molecular clone technology that the gene for IL-1Ra is 1.8 kb in length, having an open reading frame encoding 177 of amino acids; and the mature form of the IL-1Ra protein has 152 of amino acids and also has a leader sequence of 25 amino acids in length. IL-1Ra has 26%-30% homology with IL-1β, and 19% with IL-1α, with a gene structure similar to that of IL-1. Therefore, it can be postulated that the portion in which the IL-1Ra shares structural similarity with the IL-1 performs a receptor-binding function, but does not activate signal transduction across the membrane. In a pathologic state, macrophages in many tissues such as synovium and dermal tissues can produce IL-1Ra. Both the human's normal skin and the cultured keratinocytes and monocytes express IL-1Ra mRNA. Although IL-1Ra has no agonism in itself upon binding to IL-1 receptor, it can eliminate or alleviate the biological effects of the IL-1, thereby affecting pathophysiological processes in the body. The equilibrium between IL-1 and IL-1Ra determines the role of IL-1 in an inflammatory process.

Many experiments have demonstrated that IL-1Ra exerts various effects, such as suppressing generation of prostaglandins, inducing a NO concentration in the serum, reducing the amount of the expressed cyclooxygenase-2 and collagenase-1, preventing the leucocytes from infiltration and the proteoglycans in joint cartilage from degradation, antagonizing an effect of IL-1β that promote the expression of the nerve growth factor, etc., which implies a wide prospect for the use of IL-1Ra in treating inflammatory diseases such as rheumatoid arthritis, amyloidosis, osteoarthritis, allergic encephalomyelitis, etc.IL-1Ra can also improve nephritis, dermatitis and respiratory inflammation, decrease the mortality of the septic shock, increase survival rate of the heat shock, suppress growth of the myeloma, and increase the success rate of the corneal homotransplantation. Likely, some agents which can induce expression of the IL-1Ra, including the human blood serum IgA, corticosteroids, non-steroid anti-inflammatory drugs such as mofezolac, IL-4, IL-13, IFN, TGF-β, IL-6, and other cytokines performing signal transduction through gp130, are useful in treating diseases caused by IL-1.

The trade name of IL-1Ra is Kineret. Kineret (generic name Anakinra), which has been developed by Amgen, USA, is a non-glycosylated recombinant human IL-1Ra (rhIL-1Ra) having a molecular weight of 17.3 KD and consisting of 153 amino acids. The major difference between Kineret and the endogenous human IL-1Ra is that the former has one methionine residue at its N-terminus. On Nov. 14, 2001, Kineret was approved by FDA for marketing to treat adults with moderately to severely active rheumatoid arthritis who have had an absence of clinical improvement of symptoms in therapy with one or more disease-modifying anti-rheumatic drugs (DMARDs), so as to alleviate their symptoms. European Medicines Agency (EMA) has approved Kineret on Nov. 21, 2001 to be used in combination with methotrexate in patients who are not responsive adequately to treatment with methotrexate alone. At present, Kineret is under clinical trials for inflammatory bowel disease (IBD), asthma, and transplant rejection.

The onset of diabetes is associated with the impaired function of pancreatic islet β cells, and the function of pancreatic islet β cells will deteriorate progressively as pathological state extends. Currently it had been discovered that, in type 1 diabetes resulting from the destruction and the impaired functions of β cells caused by inflammation of pancreatic islet β cells, the pro-inflammatory factor IL-1β plays a key role in suppressing the functions of pancreatic islet β cells and facilitating apoptosis thereof. In patients with type 2 diabetes, it has been observed also that the expression of IL-1 in the pancreatic islet β cells is enhanced while the expression of IL-1Ra is reduced. Insufficiency of IL-1Ra appears to be a genetic property, since the genetic polymorphisms of the gene encoding IL-1Ra and the altered content of the serum IL-1Ra are correlated. IL-1β can improve the expression of the inflammatory cytokines in pancreatic islet, increase infiltration of the immune cells, leading to inflammation in the tissue, and affect the functions of β cells and the insulin sensitivity. In studies, the long-term exposure to high concentrations of glucose and a peptide hormone leptin secreted by adipose tissues would induce β cells and the pancreatic islet to produce and release IL-1β, which in turn causes the impaired functions and apoptosis of the β cells. Exogenous addition of an IL-1 receptor antagonist, such as IL-1Ra, can protect the β cells from the damage caused by high concentrations of glucose and leptin, and reduce the inflammatory marker of patients with type 2 diabetes. Similar studies also demonstrated that inflammatory mediator generated within the pancreatic islet is closely-related to diabetes (Böni-Schnetzler, et al, “Increased Interleukin (IL)-1β Messenger Ribonucleic Acid Expression in β-Cells of Individuals with Type 2 Diabetes and Regulation of IL-1β in Human Islets by Glucose and Autostimulation. ”J Clin Endocrinol Metab, 2008, 93(10): 4065-4074; Donath, et al, “Islet Inflammation Impairs the Pancreatic β-Cell in Type 2 Diabetes.” Physiology (Bethesda). 2009, 24:325-331).

In one study (Ehses et al, “IL-1 antagonism reduces hyperglycemia and tissue inflammation in the type 2 diabetic GK rat”, PNAS, 2009, 106(33):13998-14003), researchers had investigated intensively the effects of IL-1 on generation of inflammatory cytokines in pancreatic islet and inflammation in peripheral tissues responsive to insulin. GK rats, which are spontaneous, nonobese model of type 2 diabetes and develop inflammation in the pancreatic islet and the insulin resistance in peripheral tissues (liver, skeletal muscles, and adipose tissues), were chose by the researchers as the object of study. It had been found that IL-1β is expressed highly in the pancreatic islet and hepatic tissues of GK rats and that administration of IL-1Ra to the rats can block specifically the activity of IL-1 and reduce the release of inflammatory cytokines from the pancreatic islet. It was also shown in in vivo experiments that IL-1Ra can improve hyperglycemia, the function of β cells and the insulin resistance in GK rats. In addition, IL-1Ra can reduce the levels of the pancreatic islet-derived pro-inflammatory cytokines, such as, IL-1β, IL-6, TNF α, KC, MCP-1, and MIP-1 α and infiltration of CD68(+), MHC II(+), and CD53(+) immune cells into the pancreatic islet. Expression of the cytokines in hepatic tissues is also reduced. Therefore, it was concluded that IL-1Ra could improve the function of β cells and may be used in treatment of type 2 diabetes.

Marc Y. Donath et al from the University of Zurich, Switzerland, conducted a double-blind clinical trial with anakinra, and it was discovered from the results that blockage of IL-1 can improve patient's hyperglycemia and functions of pancreatic islet β cells, and lower inflammatory markers in the blood. Furthermore, Marc Y. Donath et al also performed another double-blind clinical trail with an anti-IL-1 antibody XOMA 052 to evaluate its safety and pharmacokinetics. Patients in the trail showed excellent tolerance and no severe drug-related adverse reactions were reported.

In one study, 70 patients suffering from type 2 diabetes with A₁C>7.5% and BMI>27 kg/m² were randomly assigned to undergo treatment with anakinra or placebo for 13 weeks. Thirty-nine weeks after anakinra withdrawal, it had been shown that blockage of IL-1 with anakinra can lead to improvement of the proinsulin-to-insulin (PI/I) ratio and systemic inflammatory markers compared with values in placebo-treated patients, and the improvement can last 39 weeks after treatment withdrawal (Larsen et al, “Sustained Effects of Interleukin-1 Receptor Antagonist Treatment in Type 2 Diabetes”, Diabetes Care 2009, 32: 1663-1668).

Collectively, the inflammatory cytokines are one of important causes for onset of diabetes. In addition to the conventional treatments currently used, the anti-inflammatory treatment is likely to be a novel approach of diabetic treatment.

Since IL-1 in a trace amount can exert a full biological effect, the biological effect of IL-1 can be efficaciously suppressed when the concentration of IL-1ra is usually more than 100-folds higher than that of IL-1. During the course of treating rheumatoid arthritis, the dosage of anakinra is up to 100-150 mg/d, which imposes a very high requirement on the downstream processing and manufacturing in biopharmaceuticals and is a challenge to the biopharmaceutical corporations. In theory, IL-1Ra can act on IL-1 receptors at any sites in the body with no selectivity. And it is a question whether administration of a large dosage for a long time would lead to increased infection in the patients due to immunosuppression. In particular the patients with diabetes are susceptible to infection and uneasy to be cured. Frequent dosing adds the burdens on the body, mind and economy of patients. Furthermore, IL-1Ra has an in vivo half-time of only 4-6 hours, which curtails the efficacy and increases the dosage. Consequently, the trend of new drug development is to design a novel, targeted IL-1ra, improve the efficacy in diabetic treatment, reduce unnecessary immunosuppression, reduce the dosage, and extend the in vivo time of action.

SUMMARY OF THE INVENTION

An object of the invention is to provide a novel therapeutic medicament for diabetes, the medicament comprises a fusion protein or a conjugate thereof.

In one aspect, the present invention provides a fusion protein consisting of two polypeptides which are linked together, wherein one polypeptide is an interleukin-1 receptor antagonistic protein or analogues thereof and the other polypeptide is a GLP-1 receptor binding polypeptide or analogues thereof, or a GIP receptor binding polypeptide or analogues thereof, or an insulin receptor binding polypeptide or analogues thereof.

The structure of said fusion protein is as follows: the interleukin-1 receptor antagonistic protein or an analogue thereof -L_(j)-the other polypeptide, or the other polypeptide -L_(j)-the interleukin-1 receptor antagonistic protein or an analogue thereof.

The sequence of said interleukin-1 receptor antagonistic protein or an analogue thereof is X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPH ALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVX_(IL84)ITDLSENRKQDKRFAFIRSDS GPTTSFE,SAAX_(IL116)PGWFLX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:204), wherein

X_(IL0) is a methionine, cysteine, general formula 1, general formula 2 or absent;

X_(IL66), X_(IL69), X_(IL116), or X_(IL122) is a cysteine or serine; X_(IL84) is a cysteine or asparagine;

L_(j) is a linking group or spacer group, including a long chain fatty acid, polyethylene glycol, an amino acid, a short peptide, a protein, or a long chain formed by linking one or more of the optional long chain fatty acids, polyethylene glycol, amino acids, short peptides, etc through a covalent bond, or any of the structures connecting two proteins/polypeptides through a covalent bond, or absent; preferably, said linking group or spacer group comprises one or more lysines or cysteines of which the amino or mercapto groups on the side chains are available for reacting with a modifying group; preferably, the general formula of said short peptide is a (GlyGlyGlyGlySer)_(n) (SEQ ID NO:436), wherein n is a 0, 1, 2, 3, 4, 5, or 6.

In one aspect, the present invention provides a protein conjugate which is a compound obtained through modifications such as acylation, grafting and the like on the basis of the fusion protein of the present invention.

In one aspect, the present invention provides a pharmaceutical composition comprising the fusion protein or the protein conjugate of the present invention and pharmaceutically acceptable carriers or additives.

In one aspect, the present invention provides the use of the fusion protein, the protein conjugate, or the pharmaceutical composition in treating diabetes.

In one aspect, the present invention provides the use of the fusion protein and the protein conjugate in preparing a medicament for treating diabetes.

In one aspect, the present invention provides a method of treating diabetes, the method comprising administering to a patient in need the fusion protein, the protein conjugate, or the pharmaceutical composition of the present invention.

The fusion protein and a derivative thereof has a significant efficacy in treating diabetes, and can be used at a lower dose, resulting in a marked reduction in side effects.

BRIEF DESCRIPTIONOF THE DRAWINGS

FIG. 1A and FIG. 1B are the structures of expression vectors for the native interleukin-1 receptor antagonistic protein (IL-1Ra) (IL-1ra-40-SUMO in the figure) and the G-20 (IL-1ra-SUMO in the figure) respectively.

FIG. 2 shows the DNA sequencing results of the native interleukin-1 receptor antagonistic protein (IL-1Ra) (IL-1ra-40 in the figure) and the G-20 (IL-1ra in the figure).

FIG. 3A-3C are the electrophoregrams of the fusion protein G-20 having the SUMO tag preliminarily purified by affinity chromatography.

FIG. 3A: a protein molecular weight marker is in the first lane, representing 9 K, 14 K, 22 K, 30 K, and 41 K respectively from the bottom; the cell lysate is in the second lane; the washing buffer through the column is in the third lane; the elution buffer through the column is in the fourth lane.

FIG. 3B: the fusion protein G-20 having the SUMO tag purified twice is in the first and second lanes, respectively; a protein molecular weight marker is in the third lane.

FIG. 3C: a protein molecular weight marker is in the first lane, a fusion protein G-20 having the SUMO tag removed is in the second lane.

FIG. 4 is the purity of the G-20 samples analyzed with RP-HPLC at a special buffer gradient.

FIG. 5 is the mass spectrum for the fusion protein G-20.

FIGS. 6A and 6B are the peptide mass fingerprinting for the native interleukin-1 receptor antagonist prepared by the method for protein expression in the present invention, the protein is used as a control in the animal test.

FIG. 7 is the analysis of the blood glucose in an animal test. Four groups of mice were treated with saline, the interleukin-1 receptor antagonist, the fusion proteins G-2 and G-20 (two representative fusion proteins of the GLP-1 receptor binding polypeptide—linking group-the interleukin-1 receptor antagonistic protein) respectively for 12 weeks, and then examined for the change of blood glucose in a glucose tolerance test.

FIG. 8 is the analysis of insulin level in an animal test. Four groups of mice were treated with saline (1 in the figure), the interleukin-1 receptor antagonist (2 in the figure), G-20 (3 in the figure) and G-2 (4 in the figure) (two representative fusion proteins of the GLP-1 receptor binding polypeptide- linking group-the interleukin-1 receptor antagonistic protein) for 12 weeks, and then examined for the change of insulin level in a glucose tolerance test.

FIG. 9 is the analysis of the blood glucose in an animal test. Four groups of mice were treated with saline, the interleukin-1 receptor antagonist, the fusion proteins IN-7 and IN-62 (two representative fusion proteins of the insulin receptor binding polypeptide—linking group-the interleukin-1 receptor antagonistic protein) for 12 weeks, and then examined for the change of blood glucose in an insulin tolerance test.

FIG. 10 is the analysis of blood glucose in an animal test. Four groups of mice were treated with saline, the interleukin-1 receptor antagonist, the fusion proteins GI-3 and GI-7 (two representative fusion proteins of the GIP receptor binding polypeptide- linking group-the interleukin-1 receptor antagonistic protein) for 12 weeks, and then examined for the change of blood glucose in a glucose tolerance test.

MODE OF CARRYING OUT THE INVENTION

Definition and Terms

The following definitions will apply throughout the invention unless otherwise indicated. Terms not defined herein have the meaning commonly understood in the art to which the term pertains.

In this application, a hIL-1Ra or IL-1Ra denotes the mature form of a human interleukin-1 receptor antagonist, while a IL-1ra refers to its analogues, including the interleukin-1 receptor antagonists and conjugates thereof provided in the present invention.

An “amino acid” encompasses any molecule containing both amino and carboxyl functional groups, wherein the amino and carboxyl groups are attached to the same carbon atom (the alpha carbon) in alphα-amino acid. The alpha carbon may have one or two organic substituents. An amino acid encompasses the L and D isomers and a racemic mixture. Unless otherwise specified, the amino acid residues in the sequence of the polypeptide of the present invention are all L isomers, that is, L-amino acids. And a D-amino acid is indicated by prefixing the designation of the amino acid or its abbreviation with the letter “d”, e.g., dK.

The expression “an encodable amino acid” or “an encodable amino acid residue” is used to indicate an amino acid or amino acid residue which could be encoded by a triplet (“codon”) of nucleotides, wherein

hGlu is homoglutamic acid.

α-hGlu is the L isomer of —HNCH(CO—)CH₂CH₂CH₂COOH.

δ-hGlu is the L isomer of —HNCH(COOH)CH₂CH₂CH₂CO—.

α-Asp is the L isomer of —HNCH(CO—)CH₂COOH.

β-Asp is the L isomer of —HNCH(COOH)CH₂CO—.

α-Glu is the L isomer of —HNCH(CO—)CH₂CH₂COOH.

γ-Glu is the L isomer of —HNCH(COOH)CH₂CH₂CO—.

β-Ala is —NH—CH₂—CH₂—COOH.

Sar is sarcosine.

An amino acid residue can be described with single-letter abbreviation or three-letter abbreviation.

TABLE 1 Amino Acids and abbreviations Three-letter Single-letter Three-letter Single-letter Amino acid abbreviation abbreviation Amino acid abbreviation abbreviation Glycine Gly G Threonine Thr T Alanine Ala A Cysteine Cys C Valine Val V Methionine Met M Leucine Leu L Asparagine Asn N Isoleucine Ile I Glutamine Gln Q Proline Pro P Tryptophan Trp W Phenylalanine Phe F Serine Ser S Tyrosine Tyr Y Lysine Lys K Aspartic acid Asp D Arginine Arg R Glutamic acid Glu E Histidine His H Context related to insulin:

“Native insulin” means mammalian insulin (e.g., human insulin, bovine insulin, porcine insulin etc.) from natural, synthetic, or genetically engineered sources. Human insulin comprises an A-chain of 21 amino acids and a B-chain of 30 amino acids. The two chains are linked by three disulfide bonds formed between A7 and B7, between A20 and B19, and between A6 and All. B7 and A7 indicate the amino acid residues at position 7 of the B chain (starting from the N-terminus) and at position 7 of the A chain (starting from the N-terminus) of native human insulin. Amino acid residues at other positions are denoted in a similar way.

“Insulin analogue” is a generic term of a modified insulin polypeptide, comprising a molecule of two chains consisting of an A chain and a B chain, as well as a single-chain insulin analogue, that retains high sequence homology with native insulin. “Insulin analogue” exhibits some, all or enhanced activity relative to a corresponding native insulin or is converted in vivo or into a polypeptide exhibiting some, all or enhanced activity relative to a corresponding native insulin, e.g., a polypeptide having the structure of a native insulin with one or more amino acid additions, deletions or substitutions. Proinsulins, pre-proinsulins, insulin precursors, single chain insulin precursors of humans, animals, even non-mammalian animals and analogues of any of the foregoing are also referred to herein as insulin analogues. Many insulin analogues are known in the art. Unless otherwise specified, the term “insulin analogue” herein is used broadly to include native insulin and insulin analogues.

An “insulin receptor binding polypeptide” includes insulin analogues, insulin derivatives as well as insulin conjugates, and other polypeptides which bind insulin receptor, including insulin like growth factor-1 (IGF-1), insulin like growth factor-2 (IGF-2) and their analogues, derivatives and conjugates.

Amino acids in the present application are indicated with the positions in the A chain or the B chain, unless otherwise specified. For example, A14, B28, etc. all indicate an amino acid residue or a variety thereof at the corresponding position in the A chain or the B chain of the insulin, wherein the numbering in the A chain or the B chain of the insulin starts from the first position at the N-terminus.

When an amino acid residue is individually referred to, as an example, it can be expressed as A1G, B1G, or B9H, meaning that the first amino acid in the A chain, the first amino acid in the B chain, and the ninth amino acid in the B chain is G, G, and H, respectively.

The numbering in the single-chain insulin compounds is specified in the description for the individual compounds.

A single chain insulin compound refers to a polypeptide sequence or a modified polypeptide sequence with the general structure of B chain-C_(L)-A chain, where the B chain is the B chain of insulin or an analogue thereof, the A chain is the A chain of insulin or an analogue thereof, and C_(L) is a peptidic segment connecting the C-terminus of the B chain to the N-terminus of the A chain.

GLP-1, GLP-1 analogue and GLP-1 receptor binding polypeptide: Proglucagon includes two types of glucagon-like peptides, namely GLP-1 and GLP-2.GLP-1 is mainly secreted by Langerhans cells of the terminal jejunum, ileum and colon, existing primarily in two forms : GLP-1(7-37) and GLP-1(7-36)-NH₂.GLP-1 (7-36)-NH₂ is a native form of the GLP-1 in the human body, which among the GLP-1 peptides, has the strongest effect of promoting secretion of insulin.

The sequence of the GLP-1 (7-36)-NH₂ is

(SEQ ID NO: 205) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂; the sequence of the GLP-1 (7-37) is (SEQ ID NO: 206) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG.

The in vivo expression and activities of GLP-1 are regulated tightly. After alanine at the second position of the N-terminus is hydrolyzed by a dipeptidyl peptidase (DPP-IV), an inactive GLP-1(9-36)-NH₂ is formed, which is a natural antagonist against GLP-1R in vivo. In the body, several metabolic products of GLP-1 after enzyme cleavage include GLP-1 (9-36), GLP-1 (7-35), and GLP-1 (7-34). GLP-1(9-36)-NH₂ is the major catabolic product of GLP-1 and can reach an in vivo concentration that may be higher than that of GLP-1(7-36)-NH₂ by 10 times. The in vivo half-time of GLP-1 lasts for less than 5 min with a metabolic rate of 12-13 min In physiological state, the intact GLP-1 is excreted primarily by the kidney and excluded with the aid of tissues other than the kidney.

GLP-1 are capable of facilitating insulin secretion, and the facilitating effect is glucose-dependent, the higher the blood glucose, the stronger the effect. GLP-1 binds to its receptor on the cellular membrane of the β cells in pancreatic islet and facilitates the insulin secretion by increasing intracellular cAMP, phosphorylating K⁺-ATPase, causing the K⁺ channels to be shut down, depolarization of cellular membrane, opening of Ca²⁺ channels, influx of Ca²⁺, thus stimulating the insulin secretion from cells. Such an effect is versatile, affecting various function aspects, such as transcription, translation, splicing, etc. of the pro-insulin gene. In addition, GLP-1 can also up-regulate the genes in β cells closely associated with glycol metabolism (such as glucokinase and glucose transporter-2). However, the promoter of the insulin gene can be activated partially in a PKA-independent manner

GLP-1 can also stimulate proliferation of the β cells in pancreatic islet and suppress apoptosis thereof. It has been found in studies that GLP-1 can stimulate neogenesis of the β cells in a non-STZ rat model and improve self-stabilization of the blood glucose in adult rats. In a neonatal GK rat, the constant status of blood glucose was significantly improved, the insulin level was raised, and the volume of β cells was enlarged after injection with GLP-1 or Exendin-4 for 5 days. With continuous administration of the above-mentioned medicament, the number of β cells in the rats are increased steadily, and the blood glucose controlling capability is significantly increased upon adult. It was found in culture that GLP-1 can induce differentiation of a mouse myoblast line and embryonic stem cells into the insulin-expressing cells. In a Zucker rat model of diabetes where the proportion of β cells undergoing apoptosis is up to more than 20%, treatment with GLP-1 can reduce the proportion of the apoptotic cell significantly. It has been found in studies that GLP-1 can suppress the streptozocin (STZ)-induced apoptosis of β cells in mice, while the β cells in the mice having the GLP-1R gene knocked out are significantly sensitive to the STZ-induced apoptosis.

In regulation of the blood glucose, the effect of glucagon is contrary to that of insulin in that the former increases the concentration of blood glucose by promoting glycogenolysis. It has been shown in both the healthy and patients with type 1 diabetes and type 2 diabetes that GLP-1 can suppress the release of glucagon, although the degree of suppression in the healthy is less than that in patients with diabetes. Injection of GLP-1 into a C-peptide-negative canine can decrease the plasma level of glucagon, suggesting that suppression of GLP-1 on glucagon is at least partially insulin-independent.

In a series of studies with murine, porcine and human, it was found that GLP-1 can suppress peristalsis of the gastrointestinal tract and secretion of stomach fluid and delay gastric emptying. In human being, administration of GLP-1 in both the healthy and patients with diabetes can result in temporary satiety and decreased appetite.

GLP-1 receptor (GLP-1R) is a G protein-coupled receptor with seven trans-membrane domains using cAMP as the major second messenger. It belongs to the subfamily of glucagon receptors under the B family of G protein-coupled receptors (the secretin family), of which family the most noted characteristic is an extracellular N-terminal sequence which is relatively longer and forms a spherical domain through 3 disulfide bonds. The human GLP-1 receptor has a homology of 90% with the corresponding murine receptor. The anatomic evidences demonstrate that cells with GLP-1R can be found in each of the segments of the intestinal tract in the murine, porcine and human, the distribution densities thereof vary among different species and genera, and the rule for distribution thereof is identical, that is, distribution densities of the cells with GLP-1R progressively increase from the proximal end to distal end of small intestine and the large intestine.

The amino acid sequence of the proglucagon 72-117 is referred to Bell, et al., Nature 304 368-371 (1983). The proglucagon fragment 72-108 is typically designated as GLP-1 (1-37). GLP-1(7-20) is an insulinotropic GLP-1 analogue with the shortest length as far as known.

“GLP-1 analogue” is defined as the one in which one or more of amino acid residues of GLP-1 (1-37) are deleted or substituted with other amino acid residues, or the one of which one or more of amino acid residues are inserted into the original polypeptide sequence. In a preferred embodiment, total number of amino acid residues that are different between the GLP-1 analogues and the corresponding GLP-1 (1-37) is not more than 20, or not more than 15, 10, 5, 4, 3, and 2, and most preferably is 1.GLP-1 analogues may be truncated fragments of GLP-1 (1-37).GLP-1 analogues may also be sequences obtained by extending from the N- or C-terminus of GLP-1. In certain embodiments, GLP-1 analogues comprise an extension of 1-20 of amino acids at the C-terminus. In one embodiment, the sequence extending from the C-terminus is a PSSGAPPPS-NH₂ (SEQ ID NO:438) or GPSSGAPPPS-NH₂(SEQ ID NO:437). In one embodiment, C-terminal extension comprises 1-6 of positively charged amino acids, such as arginine, lysine. In one embodiment, C-terminal extension comprises 1-6 of negatively charged amino acids, such as glutamic acid, aspartic acid. In certain embodiments, the GLP-1 analogues may be a modified product, such as a product subjected to alkyl substitution, acylation, polyethylene glycol modification, etc.

Different GLP-1s have different biological activities. Capability for GLP-1 (7-36)-NH₂ to stimulate accumulation of [¹⁴ C]-amidopyrine is higher than those of GLP-1 (1-37) and GLP-1 (1-36)-NH₂ by 100 times; GLP-1 (7-36)-NH₂ and GLP-1 (7-37)-NH₂ have comparable activities and effects. GLP-1(9-36)-NH₂ has no effect on the β cells and even acts as an antagonist against the adenylate cyclase in some studies; while it is apparent that GLP-1(7-35)-OH and GLP-1(7-34)-OH are agonists. Capability for GLP-1(7-35)-OH or GLP-1(7-37)-OH to raise the insulin level in plasma is lower than that of GLP-1(7-36)-NH₂.

The part in GLP-1 (7-36)-NH₂ which binds to the receptor is primarily the amino acid residues 7-21, although the full length sequence can play a concerted role in binding to the receptor. In addition to the skeleton of the long amino acid chain, the side chains on the amino acid residues at positions 7 (histidine), 10 (glycine), 12 (phenylalanine), 13 (threonine) and 15 (aspartic acid) are directly involved in interaction with the receptor. Furthermore, the amino acid residues at positions 28 (phenylalanine) and 29 (isoleucine) are very critical for GLP-1 to form a conformation that can be recognized by the receptor. Some studies have demonstrated that the amino acid residues at positions 10 (glycine), 15 (aspartic acid) and 17 (serine) in the sequence as well as the C-terminus of the sequence are very important for insulinotropic effect of GLP-1. For the effect of GLP-1, amidation at the C-terminus and the last amino acid residue at C-terminus are less important than the N-terminus.

The GLP-1 receptor antagonist is derivated primarily from the sequences resulting from N-terminal deletion or substitution of GLP-1 (7-36)-NH₂ or exendin-4, and comprises, but not be limited to, the following sequences:

1. (SEQ ID NO: 207) DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂; 2. (SEQ ID NO: 208) HAKGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂; 3. (SEQ ID NO: 209) EGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂; 4. (SEQ ID NO: 210) AEGTFTSEVSSYLEGQAAKEFIAWLVKGR-NH₂.

The analogues of GLP-1 include these polypeptides which are different from GLP-1 (7-37) in the amino acid sequence by 1-15 of amino acid residues, comprising substitutions or modifications at positions 11, 12, 16, 22, 23, 24, 25, 26, 27, 30, 33, 34, 35, 36, and 37. Positions 18, 20, 23, 30, 31, 34, 36, 37 or the C-terminus of these polypeptides may be linked to a modifying group, such as an albumin binding molecule or polyethylene glycol, etc.(WO2009030738).The GLP-1 receptor binding polypeptide includes any polypeptide which has a GLP-1 receptor-binding capability amounting to 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% of that of GLP-1(7-36)-NH₂, and derivatives thereof, such as GLP-1 analogues, GLP-1 receptor agonists, GLP-1 receptor antagonists, dual-agonist (coagonist) of GLP-1 and glucagon receptors, chimeric polypeptides of the GLP-1 receptor agonists and the glucagon receptor antagonist, glucagon receptor antagonists and the derivatives of these polypeptides, including the sequences in publications and literatures, such as, U.S. Pat. No. 7,235,627, WO/2002/048192, WO/2004/093823, WO/2003/103572, WO/2008/101017, WO/2009/058734, WO/2009/155258, WO/2010/070253, WO/2010/070255, WO/2011/075393, WO/2011/080102, WO/2011/073328, EP2322545, EP 2322546, “Design of a Long Acting Peptide Functioning as Both a Glucagon-like Peptide-1 Receptor Agonist and a Glucagon Receptor Antagonist, “The Journal of Biological Chemistry 2006, Vol. 281(18): 12506-12515.

With the progressive insight into GLP-1 and its correlation with diabetes, this field is becoming more focused by medicine R & D organizations at home and abroad. The representative medicaments are Exendin-4 and liraglutide.

Liraglutide has a similarity of 97% in amino acid sequence to human endogenous GLP-1 (7-37), with its sequence as follows:

(SEQ ID NO: 211) HAEGTFTSDVSSYLEGQAAK(γE-C16)EFIAWLVRGRG-OH.

Other GLP-1-like candidate agents include albiglutide, taspoglutide, lixisenatide(AVE0010 or ZP10A), etc.

Albiglutide is a GLP-1 dimer fused to human albumin, which has a resistance to DPP-IV hydrolysis and a half-time of 4-7 days.

The sequence of Lixisenatide is

(SEQ ID NO: 212) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH₂.

Taspoglutide is an amide derivative of the human GLP-1 (7-36) with the following modification: (2-methylalanine)-35-(2-methylalanine)-36-L-arginine and its sequence is

(SEQ ID NO: 213) HAibEGTFTSDVSSYLEGQAAKEFIAWLVKAibR-NH₂.

Exenatide, a synthetic product of exendin-4, consists of 39 amino acids and has a sequence homology of up to 53% with GLP-1 and the identical biological function. The sequence thereof is

(SEQ ID NO: 214) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂.

Exendin-3 is obtained from the secretory product of Heloderma horridum with the sequence

HSDGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂ (SEQ ID NO:215).

“Exendin-3 analogue” is defined as the one in which one or more of the amino acid residues of Exendin-3 are deleted or substituted with other amino acid residues, or the one of which one or more of amino acid residues are inserted into the original polypeptide sequence. The analogues of Exendin-3 include these polypeptides which are different from exendin-3 in the amino acid sequence by 1-15 amino acid residues.

“Exendin-4 analogue” is defined as the one in which one or more of the amino acid residues of Exendin-4 are deleted or substituted with other amino acid residues, or the one of which one or more of amino acid residues are inserted into the original polypeptide sequence. In a preferred embodiment, total number of amino acid residues that are different between the exendin-4 analogue and exendin-4 may be not more than 15, 10, 5, 4, 3, and 2, most preferably be 1, such as leucine 14, phenylalanine 25-exendin-4. Exendin-4 analogues may be truncated fragments of exendin-4 such as exendin-4(1-28)-NH₂, exendin-4(1-30), exendin-4(1-30)-NH₂, leucine 14, phenylalanine 25-exendin-4(1-28)-NH₂, leucine 14, alanine 22, phenyl alanine 25-exendin-4 (1-28)-NH₂.

The present invention encompasses the full length or truncated sequences of exendin-3 and exendin-4, comprising exendin-3 (1-30) or exendin-4(1-30) sequences, in which the C-terminus of these sequences may be shorten by three amino acid residues, preferably one amino acid residue; and the N-terminus thereof may be shorten by two amino acid residues, preferably one amino acid residue. Although the amino acid sequences are shortened, all these fragments of exendin have biological activity.

The present invention comprises the sequences extending from C-terminus of exendin-3 and exendin-4. In one embodiment, C-terminal extension comprises 1-6 of positively charged amino acids such as arginine, lysine. In one embodiment, C-terminal extension comprises 1-6 of negatively charged amino acids such as glutamic acid, aspartic acid. In certain embodiments, the analogues of exendin-3 and exendin-4 may be a modified product such as a product subjected to alkyl substitution, acylation, polyethylene glycol modification, etc.

GLP-1 receptor binding polypeptides with exendin-3 and exendin-4 as templates include any polypeptide and a derivative thereof which has the GLP-1 receptor-binding capability amounting to 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% of the capability of GLP-1 (7-36)-NH₂, comprising the sequences in the publications and patent documents, such as, the sequences mentioned in WO 97/46584, WO 98/05351, WO 99/25727, WO 99/25728, WO 99/07404, WO 99/40788, WO 00/41546, WO 00/41548, U.S. Pat. Nos. 7,691,963 B2, 7,407,932B2, 8,030,273B2, US 20010047084 A1, U.S. Pat. No. 5,424,286 A, WO/2013/002580, CN-200710138718.7, and CN200910135363.5, as well as in “EW, a novel recombinant analogue of exendin-4 expressed in Escherichia coli.” Scientific Research and Essays 2011 Vol. 6(14): 2941-2949, “Site-specific PEGylation of exenatide analogues markedly improved their glucoregulatory activity” British Journal of Pharmacology, 2011, 163: 399-412.

GIP and GIP Receptor Binding Polypeptide

Gastric inhibitory peptide is also known as glucose-dependent insulinotropic polypeptide, abbreviated as GIP. When the blood glucose concentration is high, GIP can regulate the blood g lucose by facilitating secretion of the insulin. Furthermore, GIP plays a key role in adipocytes and fat metabolism.

The sequence of human GIP is

(SEQ ID NO: 216) YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ.

“GIP analogue” is defined as the one in which one or more of the amino acid residues of GIP are deleted or substituted with other amino acid residues, or the one of which one or more of amino acid residues are inserted into the original polypeptide sequence. In one embodiment, GIP analogue is at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% identical in the sequence to the human GIP. In a preferred embodiment, total number of amino acid residues that are different between the GIP analogue and human GIP may be not more than 20, 15, 10, 5, 4, 3, and 2, most preferably be 1. In certain embodiments, the amino acid substitution may be at positions 1, 2, 3, 7, 10, 12, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29.GIP analogues may be truncated fragments of the GIP(1-42). In one embodiment, GIP analogues comprise the sequences obtained after removing 1-12 of the amino acid residues from C-terminus of the GIP(1-42), such as, GIP(1-38), GIP(1-39), GIP(1-30)-NH₂, etc. In one embodiment, GIP analogues comprise a polypeptide sequence of at least twelve amino acid residues starting from the N-terminus of GIP(1-42). In certain embodiments, GIP analogues comprise an extension of 1-20 of the amino acids at the C-terminus. In one embodiment, C-terminal extension comprises 1-6 of positively charged amino acids, such as arginine, lysine. In one embodiment, C-terminal extension comprises 1-6 of negatively charged amino acids, such as glutamic acid, aspartic acid. In certain embodiments, the GIP analogues may be a modified product, such as a product subjected to alkyl substitution, acylation, polyethylene glycol modification, etc.

GIP analogues and derivatives thereof may be found in a wide variety of literatures. Part of the GIP agonists can be found in the publications, such as, Salhanick, etal., Bioorg Med Chem Lett 2005, 15(18): 4114-4117; Green, etal., Diabetes 2005,7(5): 595-604; Gault, et.al., Biochem J 2002,367(Pt3): 913-920; Gault, etal., J Endocrin 2003;176: 133-141; Irwin, etal., Diabetes Obes Metab 11(6): 603-610 (epub 2009), etc. Examples of other GIP analogues are N-AcGIP(LysPAL³⁷)(Irwin, etal., “A Novel, Long-Acting Agonist of Glucose-Dependent Insulinotropic Polypeptide Suitable for Once-Daily Administration in Type 2 Diabetes”, J Pharmacol Exp Ther,2005 Vol. 314 No. 3:1187-1194), GIP(1-40), GIP(1-30)-NH₂, GIP(19-30)-NH₂, GIP(1-14)(Hinke, etal., “Identification of a bioactive domain in the amino-terminus of glucose-dependent insulinotropic polypeptide (GIP)”, Biochimica et Biophysica Acta 2001,Vol. 1547:143-155). Since the C-terminal fragment of GIP is more potent in promoting adipogenesis, partial or complete removing of the C-terminus is one of the procedures that decrease the effect in this aspect of a GIP agonist, such as GIP(1-30)-NH₂. Studies on structure-activity relationship have shown that the fragment 19-30 of GIP is critical to insulin response(“The insulinotropic region of gastric inhibitory polypeptide; fragment analysis suggests the bioactive site lies between residues 19 and 30.” Can J Physiol Pharmacol. 1996 Jan;74(1):65-72).

One determinant important for GIP activities is hydrolysis of the N-terminus of the polypeptide by DPP-4 enzyme into inactive GIP(3-42). Appropriate modification on an N-terminal residue at position 1, 2, or 3 of GIP can result in a resistance to degradation by DPP-4, even increase the biological activities of the GIP analogues. [D-Ala2]-GIP(1-42) is a super agonist to GIP receptor in a normal mouse in comparison with the native GIP, while the supernormal activity is not correlated totally with its effect on insulin in blood (“Dipeptidyl Peptidase IV-Resistant [D-Ala2]Glucose-Dependent Insulinotropic Polypeptide (GIP) Improves Glucose Tolerance in Normal and Obese Diabetic Rats”, Diabetes 2002 51: 652-661).

Removing N-terminal amino acid residues of GIP often yields GIP receptor antagonist.GIP(10-30)-NH₂, GIP(6-30)-NH₂ and GIP (7-30)-NH₂ are GIP receptor antagonists, in which the binding capacity of GIP(6-30)-NH₂ to the GIP receptor is comparable to that of GIP(1-42) (Gelling et al., “GIP(6-30 amide) contains the high affinity binding region of GIP and is a potent inhibitor of GIP1-42 action .”Regul Pept. 1997, 69(3):151-154; “Postprandial stimulation of insulin release by glucose-dependent insulinotropic polypeptide (GIP). Effect of a specific glucose-dependent insulinotropic polypeptide receptor antagonist in the rat”, J Clin Invest. 1996 Dec. 1; 98(11): 2440-2445). Other GIP antagonists include GIP(15-42), GIP(15-30) , GIP(16-30), and GIP(17-30).A GIP analogue Pro3-GIP (“Characterization of the cellular and metabolic effects of a novel enzyme-resistant antagonist of glucose-dependent insulinotropic polypeptide.” Biochem Biophys Res Commun. 2002 Feb. 8; 290(5):1420-6) antagonizes the effect of GIP in cells and in vivo in the obese diabetic ob/ob mice (“Effects of the novel (Pro(3))GIP antagonist and exendin(9-39)amide on GIP-and GLP-1-induced cyclic AMP generation, insulin secretion and postprandial insulin release in obese diabetic (ob/ob) mice: evidence that GIP is the major physiological incretin.” Diabetologia. 2003 February; 46(2):222-30; “Effects of the novel (Pro(3))GIP antagonist and exendin(9-39)amide on GIP-and GLP-1-induced cyclic AMP generation, insulin secretion and postprandial insulin release in obese diabetic (ob/ob) mice: evidence that GIP is the major physiological incretin. Diabetologia. 2003 February; 46(2):222-30). After injection with Pro3-GIP for 50 consecutive days, the adult, diabetic mice fed on a high-fat diet have decrease in body weight, in accumulation of adipose tissue, significant improvement in levels of glucose, saccharified hemoglobin and insulin, and decrease in levels of triglycerides in the muscle and the liver (McClean, etal., “GIP receptor antagonism reverses obesity, insulin resistance and associated metabolic disturbances induced in mice by prolonged consumption of high fat diet.” Am J Physiol Endocrinol Metab. 2007 December; 293(6):E1746-55).

The features of type 2 diabetes include insulin resistance and dysfunction of pancreatic islet β cells with varying degree. In European and United States, insulin resistance and obesity are major pathological characteristics of patients with diabetes. In Asia, impaired insulin secretion is the main cause. Consequently, a GIP agonist is likely to benefit patients with impaired insulin secretion, in particular patients in Asia, while GIP inhibitor is likely to benefit obesity patients with insulin resistance, in particular patients in Europe and America.

In certain embodiments, the component from which a fusion protein or dimeric protein is formed together with the interleukin-1 receptor binding polypeptide are GIP agonists, GIP inhibitors, GLP-1/GIP receptors dual-agonists, glucagon/GIP receptors dual-agonists, GLP-1/GIP/glucagon receptors tri-agonists (e.g., those mentioned in WO/2010/011439, WO2010148089 A1, US2012/0172295, US 2011/0166062, US2012/0322725), GLP-1 agonists/GIP inhibitors, or chimeric polypeptide (e.g., HG-GIP(3-30)-NH₂, HG-GIP(3-30)-exendin-4(31-39)-NH₂, Ser2-GIP(1-30)-NH₂, Ser2-GIP(1-30)-exendin-4(31-39)-NH₂, dAla2-GIP(1-30)-exendin-4(31-39)-NH₂). In the present invention, GIP receptor binding polypeptides may be any of the above-mentioned polypeptide sequences able to bind to GIP receptors. A GIP receptor binding polypeptide includes any polypeptide sequence with a binding capacity to the GIP receptor that amounts to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or higher of the binding capacity of GIP (1-42), and derivatives thereof, including GIP analogues.

GIP is effective in stimulating proliferation of (3 cells and protecting survival of β cells (“Glucose-Dependent Insulinotropic Polypeptide Is a Growth Factor for β (INS-1) Cells by Pleiotropic Signaling.” Mol Endocrinol. 2001 September; 15(9):1559-70; “Mechanisms of mitogenic and anti-apoptotic signaling by glucose-dependent insulinotropic polypeptide in beta(INS-1)-cells”, J Endocrinol. 2002 August, 174(2):233-46; “Glucose-dependent insulinotropic polypeptide promotes beta-(INS-1) cell survival via cyclic adenosine monophosphate-mediated caspase-3 inhibition and regulation of p38 mitogen-activated protein kinase”, Endocrinology. 2003 October, 144(10):4433-45).Many studies have demonstrated that GIP analogues are very potent in protecting survival in the rat model of type 2 diabetes and in INS-1β cells (“A GIP receptor agonist exhibits beta-cell anti-apoptotic actions in rat models of diabetes resulting in improved beta-cell function and glycemic control”, PLoS One. 2010 Mar. 9; 5(3):e9590; “GIP stimulation of pancreatic beta-cell survival is dependent upon phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB) signaling, inactivation of the forkhead transcription factor Foxol and downregulation of bax expression. J Biol Chem. 2005,280(23):22297-307).

Since GIP exhibits protection on the pancreatic islet β cells, a fusion protein, dimeric protein or cross-linking protein consisting of the GIP and an interleukin-1 receptor binding polypeptide would exert better effects. In the present invention, the linking pattern for the GIP receptor binding polypeptide and the interleukin-1 receptor binding polypeptide can be referred to the pattern for GLP-1 receptor binding polypeptide and the interleukin-1 receptor binding polypeptide.

Interleukin-1 Receptor Antagonistic Protein:

An “interleukin-1 receptor antagonistic protein” includes an interleukin-1 receptor antagonist (an IL-1ra) and an analogue and derivative thereof.

The family of IL-1 receptors include several receptors. Accordingly, there are several different agonists and antagonists. These antagonists do not necessarily bind to the same IL-1 receptor from the family Herein, the IL-1ra is used to stand for all of IL-1 antagonists of all receptors of the IL-1 receptor family The IL-1ra includes human IL-1Ra (hIL-1Ra), an analogue, a derivative or a functional equivalent thereof. The functional equivalent has the biological activities of hIL-1Ra, such as capable of binding to the IL-1 receptor, not producing the downstream signal transduction, and hindering the binding of IL-1 to the IL-1 receptor. Examples may be referred to U.S. Pat. Nos. 6,096,728, 6,541,623, 6,365,726, and 6,399,573.

For description of the methods for preparing the preferred IL-1ra (including glycosylated and non-glycosylated forms) and use thereof, see U.S. Pat. No. 5,075,222, WO 91/08285, WO 91/17184, AU 9173636, WO 92/16221, and WO 96/22793. Specifically, U.S. Pat. No. 5,075,222 describes three forms of IL-1Ra. The first form IL-1Raα is characterized by the molecular weight of 22-23 kD on SDS-PAGE, an isoelectric point of approximately 4.8, and elution with a Tris buffer (pH 7.6) containing approximately 52 mM NaCl from Mono Q FPLC column The second form IL-1Raβ is a protein with a molecular weight of 22-23 kD, and is eluted with a Tris buffer containing 48 mM NaCl from Mono Q FPLC column. Both the IL-1Raα and the IL-1Raβ are glycosylated. The third form IL-1Rax has the molecular weight of about 20 kD, is eluted with a Tris buffer containing 48 mM NaCl from Mono Q FPLC column, and non-glycosylated. The three antagonists have similar functions and immunological activities.

At present, it is known that IL-1Ra includes a secretory sub-type (sIL-1Ra) and three kinds of intracellular sub-type (icIL-1Ra1, 2, and 3).

The IL-1Ra set forth in the present invention may be a naturally secreted human IL-1Ra or a genetically recombinant human IL-1Ra, preferably a genetically recombinant human IL-1Ra.

In some embodiments, IL-1ra comprises anakinra and an analogue thereof. The sequence of anakinra is

(SEQ ID NO: 217) MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVV PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFA FIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQ EDE.

in which two cysteines at positions 70 and 117 can form a disulfide linkage.

The IL-1ra and an analogue thereof in the present invention may be prepared and produced by using bioengineering technologies. For convenience of expressing and producing the protein, methionine can be added at the N-terminus of the sequences of the individual proteins in the present invention if needed, without influencing the biological activity of original sequence Anakinra is the one where a methionine is added at the N-terminus of non-glycosylated hIL-1Ra.

In some embodiments, IL-1ra comprises one or more glycosylation moiety.

In the present invention, the amino acid sequence of IL-1ra can be altered without affecting the biological activity thereof. For example, one of the IL-1ra analogues may comprise one or more of conservative amino acid substitutions. The conservative amino acid substitution is the one where an amino acid residue is replaced with another amino acid residue having a similar side chain Amino acid residues can be classified according to the nature of the side chain of an amino acid residue in literatures Amino acid residues with a basic side chain comprise lysine, arginine, histidine; amino acid residues with an acidic side chain and an acylamide side chain comprise aspartic acid, glutamic acid, asparagine, glutamine; amino acid residues with small aliphatic, non-polar, or weak polar side chains comprise glycine, alanine, threonine, serine, proline; amino acid residues with large aliphatic and non-polar side chains comprise leucine, isoleucine, valine; aromatic amino acid residues comprise phenylalanine, tryptophan, tyrosine; amino acid residues with a sulphur-containing side chain comprise cysteine and methionine.

An IL-1ra analogue involved in the present invention comprises deletion of part of the amino acid residues in the amino acid sequence of IL-1ra (deletion analogue), substitutions with natural or non-natural amino acid residues (substitution analogue) or insertions (insertion analogue).The IL-1ra deletion analogue can typically have about 1-50 of amino acid residue deleted, more commonly have 1-10 of residues deleted. The IL-1ra insertion analogue may include fusion at the N-or C-terminus of IL-1ra, as well as one or more amino acid residues inserted into the internal sequence of IL-1ra. The terminal insertion of IL-1ra includes a chimeric protein. In one embodiment, the chimeric protein includes IL-1ra and the whole or parts of the constant regions in the heavy or light chain of a human immunoglobulin. The immunoglobulin portion of the preferred chimeric protein may comprise all constant regions except the first domain in the heavy chain constant regions of a human immunoglobulin (e.g., IgG, IgA, IgM, or IgE). Any amino acid residue in the immunoglobulin portions can be deleted or substituted with one or more of amino acid residues, or can be inserted with one or more of amino acid residues, so long as said IL-1ra still antagonize the IL-1 receptor, and the immunoglobulin portions exhibit one or more of their characteristic properties. The chimeric protein at the terminus of IL-1ra can also comprise the partial or whole sequence of human albumin, any amino acid residue in the albumin portion can be deleted or substituted with one or more of amino acid residues, or can be inserted with one or more of amino acid residues.

Analogues of the interleukin-1 receptor antagonistic protein set forth in the present invention is homologous approximately to IL-1Ra. The term “homologous approximately to” used herein refers to the degree of homology more than 60%, 70%, 80%, 90%, even more than 95%. The homology percentage set forth herein is calculated by an algorithm described by Dayhoff (Atlas of Protein Sequence and Structure, 5:124 (1972), National Biochemical Research Foundation, Washington, D.C.).

The IL-1ra can have a biological activity amounting to 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or higher of the biological activity of hIL-1Ra for interleukin-1 receptor.

The person skilled in the art can prepare a chemically or biologically modified IL-1ra, an analogue, and a derivative thereof. A conjugate can be prepared with a glycosylated, non-glycosylated or deglycosylated IL-1ra and an IL-1ra analogue, and a chemically modifying group used includes water-soluble polymers (e.g., polyethylene glycol) and fatty acids. One IL-1ra analogue may comprise one or more of modifying groups. The chemically modifying group is typically linked to the IL-1ra through an α- or ε-amino group of an amino acid residue or an active thiol group in the IL-1ra. An amino acid residue having free amino group includes lysine residue and an amino acid residue at the N-terminus. An amino acid residue having active thiol group includes cysteine residue.

Special substitution of an amino acid residue in the IL-1ra sequence may be of particular use, e.g., addition of a cysteine or lysine residue is favorable to linkage of modifying groups according to the embodiment method to form a conjugate. In addition, a N- or O-linked glycosylated site can be added or deleted in the sequence of IL-1ra. An asparagine-linked glycosylation recognition site includes tripeptide sequences Asn-Xaa-Ser/Thr recognized by a suitable cellular glycosylase, wherein Xaa may be any of the natural amino acids except Pro.In a mature hIL-1Ra, the asparagine at position 84 is a glycosylated site. The molecular weight of the glycosylated IL-1ra can be varied depending on the varied degree of glycosylation. The interleukin-1 receptor antagonistic protein portion in the fusion protein, dimeric protein or cross-linking protein may be linked to the modifying group (e.g., polyethylene glycol) through a mercapto group on the side chain of cysteine.

The numbering for the amino acid residues in a mature hIL-1Ra is based on the following amino acid sequence, with the position of the first amino acid residue at N-terminus being the first position:

(SEQ ID NO: 218) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE.

Four cysteines (C66, C69, C116, and C122) are present in the sequence of the above-mentioned natural IL-1ra. According to the method implemented in the present invention, the cysteine at position 116 (C116) is the special site in IL-1ra at which the modifying group (e.g., mono-polyethylene glycol) activated by maleimide or haloacetyl (e.g., I—CH₂—CO—) is reacted. In human IL-1ra, the other three cysteines is not easy to be reacted with the modifying group activated by maleimide or haloacetyl. For linking the modifying group to different sites of IL-1ra or conferring more than one modifying groups on IL-1ra, a cysteine residue can be substituted for an amino acid residue at the particular site. The IL-1ra analogues include the sequences where the cysteine residue is added at the N- or C-terminus of a protein, the sequences where the cysteine residue is substituted for the amino acid residues at original positions 6, 8, 9, 84, or 141, or the sequences where the cysteine at position 116 is substituted with a serineor one or more of the four cysteines is substituted with a serine. In addition to the IL-1ra with a single modifying group, the IL-1ra may further comprise the combination of the above-mentioned modifications, so that more than one cysteine will be reacted with the modifying groups.

The sequences of the IL-1ra analogues in the corresponding section are as follows:

(SEQ ID NO: 219) CRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVV PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFA FIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQ EDE; (SEQ ID NO: 220) CRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVV PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFA FIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQ EDE; (SEQ ID NO: 221) CRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVV PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVCITDLSENRKQDKRFA FIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQ EDE; (SEQ ID NO: 222) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVCITDLSENRKQDKRFAF IRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE; (SEQ ID NO: 223) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVCITDLSENRKQDKRFAF IRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE; (SEQ ID NO: 224) RPSGRCSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE; (SEQ ID NO: 225) RPSGRCSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE; (SEQ ID NO: 226) RPSGRKSCKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE; (SEQ ID NO: 227) RPSGRKSCKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE; (SEQ ID NO: 228) RPSGRKSSCMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE; (SEQ ID NO: 229) RPSGRKSSCMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE; or (SEQ ID NO: 230) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGCMVTKFYFQE DE.

All of these IL-1ra analogues may be used in synthesis of the fusion protein, dimeric protein or cross-linking protein of the present invention.

Modifying Moiety

Fusion proteins may contain one or more modifying moieties which provide a fusion protein, a dimeric protein, or a cross-linking protein with desired properties. For example, a modifying moiety can reduce the rate of degradation of a protein in various environments (such as the digestive tract and the blood). Preferred modifying moieties are those which permit a protein to retain a comparative binding activity to the receptor. Further, preferred modifying moieties are those which are amphiphilic or hydrophilic, or which make the modified protein more hydrophilic and less lipophilic than a corresponding unmodified protein. The modifying moieties may include degradable or hydrolizable linkages, such as lactide, glycolide, carbonate, ester, carbamate and the like. This approach allows the polymers to be degraded into lower molecular weight fragments.

The modifying moiety can include one or more hydrophilic moieties, lipophilic moieties, amphiphilic moieties, salt forming moieties, spacer moieties, linking moieties, and terminating moieties, and combinations thereof. The various moieties can be linked together by a covalent bond, or by hydrolizable or non-hydrolizable bonds. Representative hydrophilic, lipophilic and amphiphilic moieties are described in more detail below. Hydrophilic Moieties

Examples of suitable hydrophilic moieties include PAG moieties, polysaccharides, polysorbate moieties, and combinations thereof.

Polyalkylene glycol (PAG) consists of multiple alkylene glycol units. In one embodiment, the units are all identical (e.g., polyethylene glycol (PEG) or polypropylene glycol (PPG)). In another embodiment, the alkylene glycol units are different. The polymers can be random copolymers (for example, the copolymer of ethylene oxide and propylene oxide) or branched or graft copolymers.

PEG in present invention is any water soluble polyethylene glycol or polyethylene oxide. PEG has the formula —(CH₂CH₂O)_(n)—, where n is an integer from 2 to 2000. One end of PEG is usually an inert functional group, e.g., an alkyl or alkoxy etc. Although PEG capped with a methoxy group is named mPEG with the formula CH₃O(CH₂CH₂O)_(n)—, they are still generally called PEG. PEG20K is a polyethylene glycol of molecular weight 20,000.

The other end of PEG is generally an activated functional group or a functional group prone to form a covalent linkage, such as amino, carboxyl group, hydroxyl, mercapto group, aldehyde, etc. PEG-maleimide can be attached to the mercapto group on the side chain of a cysteine through Michael addition reaction; PEG-iodoacetyl (CO—CH₂—I) is reacted with the mercapto group-SH to form a stable thioether bond; PEG-NHS (succinimide) can be attached to the α-amino group of an amino acid residue or the ε-amino group on the side chain of a lysine through a nucleophilic substitution reaction (acylation); PEG-aldehyde can be attached to the amino group on a polypeptide under the action of a reductant (e.g., sodium cyano borhydride) through reductive alkylation reaction.

The commercial PEG reagents used in the invention include, but are not limited to, mPEG-SC (methyoxy-PEG-succinimidyl carbonate), mPEG-NHS (SCM) (or mPEG-SPA, succinimidyl ester of monomethoxypoly (ethylene glycol) propionic acid), NHS-PEG-NHS, mPEG-CHO (methoxy-PEG-propionaldehyde), ALD-PEG-ALD (acetaldehyde-PEG-acetaldehyde), PEG-Ts (methoxy-PEG-toluenesulfonic acid), Ts-PEG-Ts (toluenesulfonic acid-PEG-toluenesulfonic acid), mPEG-CDI (methoxy-PEG-carbonyl imidazole), CDI-PEG-CDI (carbonyl imidazole-PEG-carbonyl imidazole), mPEG-NPC (methoxy-PEG-nitrophenyl carbonate), mPEG-ISC (PEG-isocyanate), ISC-PEG-ISC (isocyanate-PEG-isocyanate), mPEG-EPO (methoxy-PEG-epoxide), EPO-PEG-EPO (epoxide-PEG-epoxide), mPEG-Mal (methoxy-PEG-maleimide), Mal-PEG-Mal (maleimide-PEG-maleimide), maleimide-PEG-NHS, maleimide-PEG-NH₂, maleimide-PEG-COOH, PEG-vinyl sulfone derivatives, (VS-PEG-X, X=NHS, maleimide, NH₂, COOH, etc), PEG-iodoacetamide derivatives (IA-PEG-X, X=NHS, maleimide, NH₂, COOH, etc.), mPEG-OPSS (methoxy-PEG-orthopyridyl disulfide), OPSS-PEG-NHS (orthopyridyldisulfide-PEG-succinimidyl ester), OPSS-PEG-NH₂, OPSS-PEG-OPSS.

The PEG in this invention can be linear, branched, forked, or dumbbell shaped. In one embodiment, a branched PEG can be represented by general formula R(-PEG-_(n)OH), in which R represents a core group (typically polyhydric) such as pentaerythritol, sugar, lysine or glycerol; m represents the number of branches, and ranges from 2 to the maximum number of attachment sites on the core group; and n represents the number of PEG, the number of PEG on each branch may be different. In another embodiment, a branched PEG can be represented by the general formula (CH₃O-PEG-_(n))_(p)R-Z, in which p equals 2 or 3, R is lysine or glycerol, Z is a reactive group. In one embodiment, forked PEG can be represented by the general formula PEG(-L-X)_(n), in which L is a linking group and X represents a terminal reactive group.

PEG can be polydisperse with a polydispersity index less than 1.05. PEG can also be monodisperse. A monodisperse PEG is composed of molecules of the same length (molecular weight) rather than a mixture of molecules of similar length (molecular weight).

The term “alkyl” comprises saturated straight or branched hydrocarbon groups.

The term “alkoxy” comprises the free radical “alkyl-O—”. The representative example is methoxy, ethoxy, propoxy (e.g., 1-propoxy and 2-propoxy), butoxy (e.g.,1-butoxy, 2-butoxy, and 2-methyl-2-propoxy), pentyloxy, hexyloxy, etc.

Saccharide Moieties

Polypeptides and proteins in this invention can contain glycosylation sites. Glycosylation can not only improve drug efficacy and pharmacokinetics of a drug, but also act in drug targeting and delivery. A saccharide moiety can be O-linked or N-linked. O-linked saccharide is generally attached to the oxygen atom of hydroxyl group in serine or threonine. N-linked saccharide is attached to the nitrogen atom of the side chain amide in asparagine. Although GLP-1 receptor binding polypeptides are not usually glycosylated, serine at the C terminal of GLP-1-PSSGAPPPS-IgG Fc (SEQ ID NO:283) can be glycosylatedand threonine(AEPKSCDKTHTCP . . . ) (SEQ ID NO:290), the 11^(st) amino acid residue at the N-terminal of Fc, can also be glycosylated.

Representative saccharide moieties include, but are not limited to, glycerol moieties, monosaccharide, disaccharide , trisaccharide , oligosaccharides, and polysaccharides such as starches, glycogen, cellulose and/or polysaccharide gums. Specific monosaccharides include C6 and above (preferably C6 to C8) saccharides such as glucose, fructose, mannose, galactose, ribose, and sedoheptulose; disaccharides and trisaccharides include moieties having two or three monosaccharide units (preferably C5 to C8) such as sucrose, cellobiose, maltose, lactose, and raffinose.

Other Hydrophilic groups

Biocompatible polycation groups include polyamines having multiple amino groups on either the backbone or the side chains, such as poly(lysine) and other polyamino acids having multiple positive charges formed by natural or synthetic amino acids, including poly(ornithine), poly(arginine), and poly(histidine), and nonpolypeptide polyamines such as poly(aminostyrene), poly(aminoacrylate), poly (N-methyl aminoacrylate), polymers of quaternary amine etc. Biocompatible polyanion groups include groups having multiple carboxyls on either the backbone or the side chains, such as poly(aspartic acid) and poly(glutamic acid) etc. Other hydrophilic groups include natural or synthetic polysaccharides such as chitosan and glucan etc.

Polyanionic Bio-Adhesives

Certain hydrophilic groups appear to have potentially useful bio-adhesive properties. Examples of such groups are found, for example, in U.S. Pat. No. 6,197,346. Those polymers containing multiple carboxyls exhibit bio-adhesive properties. Rapidly biodegradable polymers that expose multiple carboxyls upon degradation, such as poly(lactide-co-glycolide), polyanhydrides, and polyorthoesters, are also bio-adhesives. These polymers can be used to deliver polypeptide and protein drugs to the gastrointestinal tract. As the polymers degrade, they can expose carboxyl groups to enable them to adhere strongly to the gastrointestinal tract, so as to aid in the delivery of polypeptide and protein drugs.

Lipophilic Moieties

In one embodiment, the modifying moieties include one or more lipophilic moieties. The lipophilic moiety may be those well known by the skilled in the art, which include, but not limited to, alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, fatty acids, cholesteryl, lipophilic oligomers and polymers.

The hydrocarbyl can be a saturated or unsaturated, linear, branched, or cyclic hydrocarbon having one or more carbon atoms. In one embodiment, the hydrocarbyl has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more carbon atoms. The hydrocarbyl can either be unsubstituted or substituted with one or more substituents.

The lipophilic moiety may be a fatty acid, such as a natural or synthetic, saturated or unsaturated, linear or branched fatty acid. In one embodiment, the fatty acid has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more carbon atoms.

Conjugation Strategies

The degree of conjugation of a protein with modifying moieties, selection of conjugation sites and selection of modifying moieties may be varied according to actual requirements, for example, to make the conjugate less susceptible to in vivo degradation, and thus has an increased plasma half-life. A conjugation site may include an amino acid residue such as lysine. In one embodiment, protein conjugates are mono-conjugates. In another embodiment, protein conjugates are multi-conjugates. In some embodiments, protein conjugates are a mixture of mono-conjugate, di-conjugates, tri-conjugates, tetra-conjugates and the like. The modifying moieties may be the same, or different from one another. When a protein conjugate has a plurality of modifying moieties, it may be preferable to couple one or more of the modifying moieties to a fusion protein with hydrolyzable bonds and couple other modifying moieties to the fusion protein with non-hydrolyzable bonds. Alternatively, all of the bonds coupling the modifying moieties to a fusion protein may be hydrolyzable but have varying hydrolysis rates in vivo.

An ideal conjugation strategy is one in which a conjugate is endowed with some or all biological activities of the original protein. The preferred conjugation sites of an insulin receptor binding polypeptide includes B1 (N-terminus) of a double chain insulin analogue, the amino group on the side chain of a lysine or the mercapto group on the side chain of a cysteine originally present or introduced after substitution or insertion at the C-terminus of B chain, the amino group on the side chain of a lysine or the mercapto group on the side chain of a cysteine introduced after substitution or insertion at the C-terminus of A chain, the amino group on the side chain of a lysine or the mercapto group on the side chain of a cysteine originally present or introduced after substitution or insertion at the N-or C-terminus of a single chain insulin analogue. A B1 mono-conjugate and a di-conjugate of the B chain are most commonly used. Furthermore, other conjugation sites can be created by embedding a natural or non-natural amino acid having an amino or mercapto group into the C peptide linker, or the A chain, the B chain of a single chain insulin analogue. The conjugation sites used most commonly in an interleukin-1 receptor binding polypeptide include the mercapto group on the side chain of a cysteine at positions 84 and 116 or the amino group at the N-terminus. The conjugation sites used most commonly in a GLP-1 analogue include the amino group on the side chain of a lysine or the mercapto group on the side chain of a cysteine at positions 10, 20, and 28 starting from N-terminus of GLP-1(7-37)-OH or GLP-1(7-36)-NH₂, originally present or introduced after substitution or insertion, or the amino group on the side chain of a lysine or the mercapto group on the side chain of a cysteine introduced after substitution or insertion at the C-terminus .The conjugation sites used most commonly in an Exendin-4 analogue include the amino group on the side chain of a lysine or the mercapto group on the side chain of a cysteine at positions 12, 20, 27, 28 and 32 starting from N-terminus of Exendin-4, originally present or introduced after substitution or insertion, or the amino group on the side chain of a lysine or the mercapto group on the side chain of a cysteine substituted or inserted at the C-terminus.

A modifying moiety may be coupled to a protein with a hydrolyzable bond (e.g., an ester, carbonate or hydrolyzable carbamate bond). Use of a hydrolyzable bond will allow a protein conjugate to act as a prodrug. For example, when a modifying moiety conjugation site is in a binding region of a protein to the receptor, the conjugate of the protein and modifying moiety lacks activity. The active protein is released as one or more modifying moieties are detached from the conjugate. Use of a hydrolyzable bond can also provide a delayed-release or controlled-release effect.

In one example, a protein is coupled to a modifying moiety by a non-hydrolyzable bond (such as amide bond and ether bond). And the non-hydrolyzable bond may contribute to the prolongation of circulation time for protein conjugate in the plasma if necessary.

A protein, protein analogue, or protein derivative may be coupled to the modifying moiety by a various nucleophilic functional groups, including, but not limited to, nucleophilic hydroxyl or amino Nucleophilic hydroxyl may be found, for example, in serine, threonine, or tyrosine, and nucleophilic amino may be found, for example, in histidine, Lysine or at the N-terminus of the A or B chains of insulin or insulin analogue. A protein may be coupled to a modifying moiety by a free —SH group, e.g., by forming a thioester, thioether or sulfonate bond.

One of the reasons for short circulation time of small molecular weight polypeptides or proteins in the plasma is renal clearance. Increasing the molecular weight of polypeptides or proteins to the critical point for renal clearance of over 40,000 daltons will significantly reduce the renal clearance rate and prolong duration of action of the polypeptides. One common approach is to conjugate a protein or polypeptide to a natural or synthetic large molecule via a hydrolysable or non-hydrolysable bond. Biomacromolecules include albumin, polysaccharides (such as glucan), and antibody (such as IgG) etc. Albumin and IgG account for 90% of plasma proteins and have weeks of in vivo circulation time. Polypeptides conjugated to albumin or IgG Fc display a markedly longer in vivo circulation time. For example, etanercept is a product of soluble TNF2 receptor bound to the IgG1 Fc, such a receptor dimer with a 150 kDa molecular weight is an effective antiinflammatory agent which is administered twice weekly(t_(1/2)=102 hours). Recently approved romiplostim for stimulation of platelet production is another example of IgG fusion protein.

The albumin may be human albumin (HSA), an analogue of human albumin or a portion of human albumin. Human albumin comprises 585 of amino acid residues with a molecular weight of 66500.EP322,094 provides shorter sequences of human albumin, including HAS (1-373), HAS (1-388), HAS (1-389), HAS (1-369), HAS (1-419), and the fragment between amino acid positions 1-369 and 1-419. The sequences provided in EP399666 include HAS (1-177), HAS (1-200), and the fragment between HSA(1-177) and HAS (1-200). The Fc portion of an immunoglobulin may be the Fc portion of a human immunoglobulin, an analogue of the Fc portion of human immunoglobulin, a fragment of the Fc portion of human immunoglobulin, etc. Fc may comprise the hinge-region, C_(H2), C_(H3) up to the C-terminus. A major reason for the Fc fusion protein to extend the in vivo circulation time is that the Fc is able to bind to FcRn. FcRn distributes over the surface of endothelial cells, binds to IgG in a pH-dependent manner, and protects IgG from degradation. Variations at the interface between CH₂ and CH₃ may extend the half-time of IgG (Hinton PR, et al., 2004. Engineered human IgG antibodies with longer serum half-lives in primates. J Biol Chem. 279(8):6213-6; Vaccaro C., et al., 2005. Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels. Nat Biotechnol. 23(10):1283-8). As 70% of albumins in the blood vessel are mercaptalbumin, the mercapto group on the side chain of the cysteine-34 is the most active mercapto group in the blood plasma. The inventive protein, analogue, or derivative thereof may be reacted with it by a linking group with an active functional group at one of its ends such as maleimide, etc., to generate a fusion protein-albumin conjugate. In one embodiment, the N-or C-terminus of the protein in the present invention can be linked to the N-or C-terminus of the albumin or immunoglobulin Fc. This linking strategy enables the circulation time of small molecule weight compound to reach the half-time of plasma protein to which it binds, but at the price of possible decrease in the receptor binding affinity. One of the solutions to the above is insertion of a spacer group between the polypeptide (or the protein) and the modifying group. The spacer group, such as β-alanine, γ-aminobutyric acid, γ-glutamic acid or polyethylene glycol, may be used between an amino group of the polypeptide/protein and a modifying group. Palmitic acid group of Liraglutide is linked to the side chain of a lysine through a spacer group. An additional solution is that the modifying group is linked to the polypeptide/protein through a “reversible” linking group.

The Fusion Protein/Dimeric Protein/Cross-Linking Protein

The fusion protein, dimeric protein or cross-linking protein referred in the present invention is a product obtained by linking a GLP-1 receptor binding polypeptide, a GIP receptor binding polypeptide or an insulin receptor binding polypeptide to an interleukin-1 receptor antagonistic protein. The two portions of the polypeptide/protein can be directly linked and can also be linked through a linking group (or a spacer group). The fusion protein/dimeric protein/cross-linking protein can have a biological activity for an interleukin-1 receptor amounting to 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or higher of the biological activity of IL-1RA, while as to the other portion, the GLP-1 receptor binding polypeptide, the GIP receptor binding polypeptide, or the insulin receptor binding polypeptide has a biological activity for a GLP-1 receptor, a GIP receptor, or an insulin receptor, respectively amounting to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or higher of the biological activity of GLP-1 (7-36)-NH₂, GIP (1-42), or the human insulin, respectively.

The fusion protein, dimeric protein, cross-linking protein and derivatives thereof in the present invention may be prepared and produced by using bioengineering technologies. For convenience of expressing and producing the protein, methionine can be added at the N-terminus of the sequences of the individual proteins of the present invention according to the requirements.

The linking group (or the spacer group) may be chemically synthesized molecules, such as long-chain fatty acids or polyethylene glycol, and may also be a natural or non-natural amino acid (e.g., lysine, glutamic acid, aspartic acid), a short peptide (e.g., β-alanine-β-alanine), a protein, or a long chain formed by one or more optional long-chain fatty acids, polyethylene glycol, amino acids, short peptides, etc linked together through covalent bonds, or any structure connecting two active polypeptides/proteins through a covalent bond, or absent.

Through a chemical reaction, the linking group may connect two polypeptides/proteins originally not attached (one is an interleukin-1 receptor antagonistic protein, the other is a GLP-1 receptor binding polypeptide, a GIP receptor binding polypeptide, or an insulin receptor binding polypeptide). In one embodiment, the linking group may have two activating groups (e.g., N-hydroxyl succinimide (NHS ester) or maleimide), each of which is reacted with the amino or mercapto group on the interleukin-1 receptor antagonistic protein and the GLP-1 receptor binding polypeptide (or the GIP receptor binding polypeptide or the insulin receptor binding polypeptide), linking the three components as a whole. In one embodiment, the linking group may have an activating group (e.g., NHS ester, maleimide, iodoacetyl, or vinylsulfone) and a functional group (e.g., an amino or mercapto group), each of which is reacted respectively with a functional group (e.g., an amino or mercapto group) and an activating group (e.g., NHS ester, maleimide, iodoacetyl, or vinylsulfone) on the interleukin-1 receptor antagonistic protein and the GLP-1 receptor binding polypeptide (or the GIP receptor binding polypeptide or the insulin receptor binding polypeptide), linking the three components as a whole. In another embodiment, the linking group may have two functional groups (e.g., an amino or mercapto group), each of which is reacted with the activating groups (e.g., NHS, maleimide, iodoacetyl, or vinylsulfone) on the interleukin-1 receptor antagonistic protein and the GLP-1 receptor binding polypeptide (or the GIP receptor binding polypeptide or the insulin receptor binding polypeptide), linking the three components as a whole. The positions of activating group and functional group needs to be determined according to the requirement for a particular reaction.

If the linking group is already linked to a biologically active polypeptide (e.g., an interleukin-1 receptor antagonistic protein) and is to be linked through a chemical reaction to another biologically active polypeptide originally not attached, the reaction mode is similar to the above. For connection of the linking group already linked to a polypeptide to another polypeptide originally not attached, one entity is required to have a functional group (e.g., an amino or mercapto group) and the other is required to have a reactive group (e.g., NHS ester, maleimide, iodoacetyl, or vinylsulfone). As for location of the reactive group and the functional group in the linking group or the polypeptide, it needs to be determined according to the requirement from a particular reaction.

In one particular embodiment, the linking group (or the spacer group) comprises one or more lysine or cysteine residues, of which the amino or mercapto groups on the side chains are used to be reacted with the modifying group. In one embodiment, the GLP-1 receptor binding polypeptide, the GIP receptor binding polypeptide or the insulin receptor binding polypeptide is linked to the interleukin-1 receptor antagonistic protein through a short peptide, forming a single-chain fusion protein. The general formula for the short peptide is (GlyGlyGlyGlySer)_(n) (SEQ ID NO:436), wherein n is a 0, 1, 2, 3, 4, 5, or 6.

1. the fusion protein consisting of the GLP-1 receptor binding polypeptide and the interleukin-1 receptor antagonistic protein

In one embodiment, the sequence of the GLP-1 receptor binding polypeptide on the basis of the human GLP-1 is

-   X_(G1)X_(G2)X_(G3)GX_(G5)X_(G6)TSDX_(G10)SX_(G12)YLEX_(G16)X_(G17)X_(G18)AX_(G20)X_(G21)FIX_(G24)X_(G25)LX_(G27)X_(G28)X_(G29)     X_(G30)X_(G31) (SEQ ID NO:231), wherein -   X_(G1) is a histidine, D-histidine, deamino-histidine,     β-hydroxyl-histidine, homohistidine, α-fluoromethyl-histidine,     α-methyl-histidine, N-methyl-histidine, N^(α)-acetyl-histidine,     α-methyl-histidine, 2-pyridinyl-alanine, 3-pyridinyl-alanine,     4-pyridinyl-alanine, imidazopropionyl; X_(G2) is a 2-methylalanine     (Aib), glycine, D-serine, serine, threonine, leucine, isoleucine,     alanine, valine, aminocyclopropanecarboxylic acid,     aminocyclobutanecarboxylic acid, aminocyclopentanec arboxylic acid,     aminocyclohexanecarboxylic acid, aminocycloheptanecarboxylic acid,     or aminocyclooctanecarboxylic acid; X_(G3) is a glutamic acid,     aspartic acid, or glutamine; X_(G5) is a threonine, aspartic acid,     glutamic acid, arginine, alanine, lysine, or histidine; X_(G6) is a     phenylalanine, tyrosine, tryptophan, or histidine; X_(G 10) is a     valine, tyrosine, phenylalanine, tryptophan, histidine, leucine,     isoleucine, serine, threonine, aspartic acid, glutamic acid,     alanine, lysine, arginine, cysteine, general formula 1, or general     formula 2; X_(G12) is a serine, isoleucine, lysine, arginine,     cysteine, general formula 1, or general formula 2; X_(G16) is a     glycine, serine, histidine, glutamic acid, aspartic acid, glutamine,     asparagine, lysine, arginine, homoglutamic acid, cysteine,     homocysteine, or cysteic acid; X_(G17) is a glutamine, arginine,     isoleucine, glutamic acid, aspartic acid, histidine, lysine,     cysteine, general formula 1, or general formula 2; X_(G18) is an     alanine, arginine, histidine, glutamic acid, or lysine; X_(G20) is     lysine, arginine, aspartic acid, glutamic acid, glutamine,     histidine, 2-methylalanine, cysteine, general formula 1, or general     formula 2; X_(G21) is an aspartic acid, glutamic acid, leucine,     alanine, lysine, cysteine, general formula 1, or general formula 2;     X_(G24) is an alanine, glutamine, asparagine, glutamic acid,     aspartic acid, serine, or histidine; X_(G25) is an alanine,     tryptophan, phenylalanine, tyrosine, cysteine, lysine, general     formula 1, or general formula 2; X_(G27) is a valine, leucine,     lysine, arginine, alanine, glycine, cysteine, general formula 1, or     general formula 2; X_(G28) is a lysine, arginine, aspartic acid,     glutamic acid, alanine, asparagine, cysteine, general formula 1, or     general formula 2; X_(G29) is a glycine, glutamine, threonine,     serine, lysine, arginine, cysteine, general formula 1, or general     formula 2; X_(G30) is an arginine, lysine, glycine, histidine,     cysteine, general formula 1, or general formula 2; X_(G31) is a     —NH₂, glycine, threonine, serine, aspartic acid, glutamic acid,     tryptophan, tyrosine, phenylalanine, histidine, arginine, lysine,     cysteine, a linking or spacer group, general formula 3, general     formula 4 or a linking or spacer group +X_(G32), in which X_(G32) is     a lysine, cysteine, general formula 3, general formula 4 or absent;     in one particular embodiment, X_(G31) is GPSSGAPPPS (SEQ ID NO:437)     or PSSGAPPPS (SEQ ID NO:438), in which any one of the amino acid     residues can be substituted or deleted or is a general formula 1, or     general formula 2, or general formula 3, or general formula 4;

In the present invention, the structure of general formula 1 is

in which J is the structure of Ln-ML, the structure of -W-X-Y-Z or a hydrogen atom;

ML is a modifying group, including, but not limited to, -W-X-Y-Z, a fatty acid, polyethylene glycol, albumin, IgG Fc, glycosyl, etc;

Ln is a optional linking group, covalent linkage or absent; the optional linking group includes, but are not limited to, polyethylene glycol, a long chain fatty acid, a polypeptide, a natural or non-natural amino acid, or a long chain formed by linking one or more of polyethylene glycol molecules, fatty acids, polypeptides, amino acid molecules through covalent bonds. In one embodiment, Ln may be a —NH—(CH₂CH₂O)_(n)—CH₂—CO—, —NH—(CH₂)_(n)—CO—, —NH—(CH₂CH₂O)_(r)—(CH₂)_(r)—CO—, n is an integer from 1 to 25, and r is an integer from 1 to 16. In one particular embodiment, L_(n) is a —NH—(CH₂CH₂O)₂—CH₂—CONH—(CH₂CH₂O)₂—CH₂—CO—. In one embodiment, L_(n) is a —NH—(CH₂)_(n1)—O—(CH₂CH₂O)_(n2)—(CH₂)_(n3)—CO—, n1, n2, and n3 are each an integer from 1 to 18. In one embodiment, L_(n) is a —NH—(CH₂)_(n1)—(OCH₂CH₂)_(n2)—CO—, n1 and n2 are each an integer from 1 to 18. In the above embodiments, L_(n) is involved in formation of an amide bond between the carbon atom of the underlined carbonyl and an amino group on a side chain of a polypeptide, and the other terminus thereof forms a covalent linkage with M_(L). In one embodiment, L_(n) is involved in formation of an amide bond between the carbon atom of the underlined carbonyl and an amino group on a side chain of a polypeptide, and the other terminus thereof forms a covalent linkage with -W-X-Y-Z.

In the present invention, the structure of -W-X-Y-Z is W is an α-amino acid residue having a carboxyl group in the side chain, this residue forms, with one of its carboxyl groups, an amide group together with the α-amino group of the N-terminal amino acid residue of the polypeptide/protein, or together with the ε-amino group of a Lys residue present in the polypeptide/protein, or together with the amino group at the end of L_(n);

Or W is a chain composed of two, three or four α-amino acids linked together via amide bonds, this chain linked via an amide bond is linked to the α-amino group of the N-terminal amino acid residue of the polypeptide or protein, or to the ε-amino group of a Lys residue present in the polypeptide or protein, or to the amino group at the end of L_(n), the amino acid residues of W being selected from the group of amino acid residues having a neutral side chain and/or amino acid residues having a carboxyl group in the side chain so that W has at least one amino acid residue which has a carboxyl group in the side chain; or

Or W is a covalent bond from X to the α-amino group of the N-terminal amino acid residue of the polypeptide or protein, or to the ε-amino group of a Lys residue present in the polypeptide or protein, or to the amino group at the end of L_(n);

X is —CO—, —CH(COOH)CO—, —N(CH₂COOH)CH₂ CO—, —N(CH₂COOH)CH₂CON—(CH₂COOH)CH₂CO—, —N(CH₂CH₂COOH)CH₂CH₂CO—, —NHCH(COOH)(CH₂)₄NHCO—, —N(CH₂CH₂COOH)CH₂CH₂CON(CH₂CH₂COOH)CH₂CH₂ CO—, —N(CH₂CH₂COOH)CH₂ CO— or —N—(CH₂COOH)CH₂CH₂ CO—, wherein

a) the above-mentioned X forms an amide bond with an amino group in W through the bond of the underlined carbonyl C, when W is an amino acid residue or a series of amino acid residues, or

b) the above-mentioned X forms an amide bond with the α-amino group of an amino acid residue at the N-terminus of the polypeptide/protein, or the ε-amino group of a Lys residue in the polypeptide/protein, or the amino group at the end of L_(n) through the bond of the underlined carbonyl C, when W is a covalent bond;

Y is —(CH₂)m, wherein m is an integer from 6 to 32;

or a divalent hydrocarbon chain comprising 1, 2 or 3 of —CH═CH-groups and multiple of —CH₂-groups, wherein the number of —CH₂-groups allows the total number of carbon atoms in the chain to reach a range of 10 to 32;

or a divalent hydrocarbon chain of the general formula —(CH₂)_(v)C₆H₄(CH₂)w-, wherein v and w are integers or one of them is zero so that the sum of v and w is in the range of 6 to 30; and

Z is —COOH; —CO-Asp; —CO-Glu; —CO-Gly; —CO-Sar; —CH(COOH)₂; —N(CH₂COOH)₂; —SO₃H; —PO₃H; or absent, provided that Z is not —COOH when W is a covalent bond and X is —CO—.

W in the side chain -W-X-Y-Z can be a covalent bond. Alternatively, W can be an α-amino acid residue having a carboxyl group in its side chain and comprising a total of from 4 to 10 carbon atoms. W also can be an α-amino acid residue encoded by a genetic code. For example, W can be selected from the group consisting of α-Asp, β-Asp, α-Glu, and γ-Glu. Further options for W are for example α-hGlu and δ-hGlu.

In a further embodiment, W is a chain composed of two α-amino acid residues of which one has from 4 to 10 carbon atoms and a carboxyl group in the side chain while the other has from 2 to 11 carbon atoms but no free carboxyl group. The α-amino acid residue with no free carboxyl group can be a neutral, encodable α-amino acid residue. Examples of W according to this embodiment are: α-Asp-Gly; Gly-α-Asp; β-Asp-Gly; Gly-β-Asp; α-Glu-Gly; Gly-α-Glu; γ-Glu-Gly; Gly-γ-Glu; α-hGlu-Gly; Gly-α-hGlu; δ-hGlu-Gly; and Gly-δ-hGlu.

In a further embodiment, W is a chain composed of two α-amino acid residues, independently having from 4 to 10 carbon atoms, and both having a carboxyl group in the side chain. One of these α-amino acid residues or both of them can be encodable α-amino acid residues. Examples of W according to this embodiment are: α-Asp-α-Asp; α-Asp-α-Glu; α-Asp-α-hGlu; α-Asp-β-Asp; α-Asp-γ-Glu; α-Asp-δ-hGlu; β-Asp-α-Asp; β-Asp-α-Glu; β-Asp-α-hGlu; β-Asp-β-Asp; β-Asp-γ-Glu; β-Asp-δ-hGlu; α-Glu-α-Asp; α-Glu-α-Glu; α-Glu-α-hGlu; α-Glu-β-Asp; α-Glu-γ-Glu; α-Glu-δ-hGlu; γ-Glu-α-Asp; γ-Glu-α-Glu; γ-Glu-α-hGlu; Y-Glu-β-Asp; γ-Glu-γ-Glu; γ-Glu-δ-hGlu; α-hGlu-α-Asp; α-hGlu-α-Glu; α-hGlu-α-hGlu; α-hGlu-β-Asp; α-hGlu-γ-Glu; α-hGlu-δ-hGlu; δ-hGlu-α-Asp; δ-hGlu-α-Glu; δ-hGlu-α-hGlu; δ-hGlu-β-Asp; δ-hGlu-γ-Glu; and δ-hGlu-δ-hGlu.

In a further embodiment, W is a chain composed of three α-amino acid residues, independently having from 4 to 10 carbon atoms, the amino acid residues of the chain being selected from the group of residues having a neutral side chain and residues having a carboxyl group in the side chain so that the chain has at least one residue which has a carboxyl group in the side chain. In one embodiment, the amino acid residues are encodable residues.

In a further embodiment, W is a chain composed of four α-amino acid residues, independently having from 4 to 10 carbon atoms, the amino acid residues of the chain being selected from the group of residues having a neutral side chain and residues having a carboxyl group in the side chain so that the chain has at least one residue which has a carboxyl group in the side chain. In one embodiment, the amino acid residues are encodable residues.

In one embodiment, W in -W-X-Y-Z can be connected to the ε-amino group of the Lys residue via a urea derivative.

The X of the side chain -W-X-Y-Z can be a group of the general formula —CO—, which via a bond from the underlined carbonyl carbon, forms an amide bond with an amino group in W; or when W is a covalent bond, via a bond from the underlined carbonyl carbon, X forms an amide bond with the N-terminal α-amino group in the polypeptide/protein, or with the ε-amino group of a Lys residue present in the polypeptide/protein, or with amino group at the end of L_(n).

In a further embodiment, the X of the side chain -W-X-Y-Z can be a group of the general formula —CH(COOH)CO—, which via a bond from the underlined carbonyl carbon, forms an amide bond with an amino group in W or; or when W is a covalent bond, via a bond from the underlined carbonyl carbon, X forms an amide bond with the N-terminal α-amino group in the polypeptide/protein, or with the ε-amino group of a Lys residue present in the polypeptide/protein, or with amino group at the end of L_(n).

In a further embodiment, the X of the side chain -W-X-Y-Z can be a group of the general formula —N(CH₂COOH)CH₂ CO—, which via a bond from the underlined carbonyl carbon, forms an amide bond with an amino group in W or; or when W is a covalent bond, via a bond from the underlined carbonyl carbon, X forms an amide bond with the N-terminal α-amino group in the polypeptide/protein, or with the ε-amino group of a Lys residue present in the polypeptide/protein, or with amino group at the end of L_(n).

In a further embodiment, the X of the side chain -W-X-Y-Z can be a group of the general formula —N(CH₂CH₂COOH)CH₂ CO—, which via a bond from the underlined carbonyl carbon, forms an amide bond with an amino group in W or; or when W is a covalent bond, via a bond from the underlined carbonyl carbon, X forms an amide bond with the N-terminal α-amino group in the polypeptide/protein, or with the ε-amino group of a Lys residue present in the polypeptide/protein, or with amino group at the end of L_(n).

In a further embodiment, the X of the side chain -W-X-Y-Z can be a group of the general —ormula -N(CH₂COOH)CH₂CH₂ CO—, which via a bond from the underlined carbonyl carbon, forms an amide bond with an amino group in W or; or when W is a covalent bond, via a bond from the underlined carbonyl carbon, X forms an amide bond with the N-terminal α-amino group in the polypeptide/protein, or with the ε-amino group of a Lys residue present in the polypeptide/protein, or with amino group at the end of L_(n).

In a further embodiment, the X of the side chain -W-X-Y-Z can be a group of the general formula —N(CH₂COOH) CH₂CON(CH₂COOH)CH₂ CO—, which via a bond from the underlined carbonyl carbon, forms an amide bond with an amino group in W or; or when W is a covalent bond, via a bond from the underlined carbonyl carbon, X forms an amide bond with the N-terminal α-amino group in the polypeptide/protein, or with the ε-amino group of a Lys residue present in the polypeptide/protein, or with amino group at the end of L_(n).

In a further embodiment, the X of the side chain -W-X-Y-Z can be a group of the general formula —N(CH₂CH₂COOH)CH₂CH₂ CO—, which via a bond from the underlined carbonyl carbon, forms an amide bond with an amino group in W or; or when W is a covalent bond, via a bond from the underlined carbonyl carbon, X forms an amide bond with the N-terminal α-amino group in the polypeptide/protein, or with the ε-amino group of a Lys residue present in the polypeptide/protein, or with amino group at the end of L_(n).

In a further embodiment, the X of the side chain -W-X-Y-Z can be a group of the general formula —N(CH₂CH₂COOH) CH₂CH₂CO N(CH₂CH₂COOH) CH₂CH₂ CO—, which via a bond from the underlined carbonyl carbon, forms an amide bond with an amino group in W or; or when W is a covalent bond, via a bond from the underlined carbonyl carbon, X forms an amide bond with the N-terminal α-amino group in the polypeptide/protein, or with the ε-amino group of a Lys residue present in the polypeptide/protein, or with amino group at the end of L_(n).

The Y of the side chain -W-X-Y-Z can be a group of the general formula —(CH₂)_(m)—, where m is an integer in the range from 6 to 32, from 8 to 20, from 12 to 20, or from 12-16.

In another embodiment, Y of the side chain -W-X-Y-Z is a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH-groups and a number of —CH₂-groups sufficient to give a total number of carbon atoms in the chain in the range of from 6 to 32, from 10 to 32, from 12 to 20, or from 12-16.

In another embodiment, Y of the side chain -W-X-Y-Z is a divalent hydrocarbon chain of the general formula —(CH₂)_(v)C₆H₄(CH₂)_(w)—, wherein v and w are integers or one of them is zero so that the sum of v and w is in the range of from 6 to 30, from 10 to 20, or from 12-16.

In one embodiment, the Z of the side chain -W-X-Y-Z is —COOH, provided that when W is a covalent bond and X is —CO—, then Z is not —COOH.

In another embodiment, the Z of the side chain -W-X-Y-Z is —CO-Asp, —CO-Glu, —CO-Gly, —CO-Sar, —CH(COOH)₂, —N(CH₂COOH)₂, —SO₃H or —PO₃H;

In a further embodiment, W of the side chain -W-X-Y-Z is selected from the group consisting of α-Asp, β-Asp, α-Glu, and γ-Glu; X is —CO— or —CH(COOH)CO; Y is —(CH₂)_(m)—, where m is an integer in the range of 12-18 and Z is —COOH, —CH(COOH)₂ or absent;

In a further embodiment, W of the side chain -W-X-Y-Z is selected from the group consisting of α-Asp, β-Asp, α-Glu or γ-Glu; -X-Y-Z is —CO(CH₂)_(n)CH₃, which via a bond from the underlined carbonyl carbon, forms an amide bond with an amino group in W; n is an integer in the range of 10-20.

In a further embodiment, W of the side chain -W-X-Y-Z is selected from the group consisting of α-Asp, β-Asp, α-Glu or γ-Glu; -X-Y-Z is —CO(CH₂)₁₂CH₃.

In a further embodiment, W of the side chain -W-X-Y-Z is selected from the group consisting of α-Asp, β-Asp, α-Glu or γ-Glu; -X-Y-Z is —CO(CH₂)₁₄CH₃.

In a further embodiment, W of the side chain -W-X-Y-Z is selected from the group consisting of α-Asp, β-Asp, α-Glu or γ-Glu; -X-Y-Z is —CO(CH₂)₁₆CH₃.

In the present invention, the structure of general formula 2 is

wherein the basic structure of O_(L) is M_(r)-L_(r)-NH-J, M_(r)-L_(r)-Z₁, M_(r), M_(r)-L_(r)-M_(L), or a hydrogen atom;

J and M_(L) are defined as the above in the present invention;

M_(r) is a functional group capable of reacting with the mercapto group to form a covalent linkage, for example, maleimide, vinylsulfone, iodoacetyl, etc.; L_(r) is an alternative linking group, covalent linkage or absent, including, but not limited to, polyethylene glycol, a long chain fatty acid, or a long chain formed by linking together one or more polyethylene glycol molecules and long chain fatty acid molecules through covalent bonds.

The two M_(r) at ends of L_(r) in M_(r)-L_(r)-M_(r) can be identical or different; a covalent linkage is formed after M_(r) at one end of M_(r)-L_(r)-M_(r) is reacted with an IL-1ra.

When M_(L) is an immunoglobulin or IgG Fc, one end of the linking group is M_(r) and linked to the IL-1ra while the other end is an aldehyde group which is reacted with the amino group on an immunoglobulin or the Fc of an IgG through reductive amination to form a covalent linkage. M_(L) includes part of or the whole of constant regions of the heavy or light chain of a human immunoglobulin. The immunoglobulin portion may include all constant regions except the first domain in the heavy chain constant regions of a human immunoglobulin (e.g., IgG, IgA, IgM, or IgE). Any amino acid residue in each of the immunoglobulin portions can be deleted or substituted with one or more of amino acid residues, or can be inserted with one or more of amino acid residues.

O_(L) includes, but is not limited to the following structure:

—CH₂-CONH—(CH₂)_(n)—NH-J, a thioether bond is formed by a bond from the underlined carbon atom and the sulphur atom of a cysteine;

—CH₂—CONH—(CH₂)_(n)—Z₁, a thioether bond is formed by a bond from the underlined carbon atom and the sulphur atom of a cysteine;

—CH₂—CONH—(CH₂CH₂O)_(n)—(CH₂)_(m)—NH-J, a thioether bond is formed by a bond from the underlined carbon atom and the sulphur atom of a cysteine;

—CH₂—CONH—(CH₂CH₂O)_(n)—(CH₂)_(m)—Z₁, a thioether bond is formed by a bond from the underlined carbon atom and the sulphur atom of a cysteine;

—CH₂—CH₂—SO₂—(CH₂CH₂O)_(n)—(CH₂)_(m)—NH-J, a thioether bond is formed by a bond from the underlined carbon atom and the sulphur atom of a cysteine;

—CH₂—CH₂-SO₂—(CH₂CH₂O)_(n)—(CH₂)_(m)—Z₁, a thioether bond is formed by a bond from the underlined carbon atom and the sulphur atom of a cysteine;

in which Z₁ is —COOH, —CO-Asp, —CO-Glu, —CO-Gly, —CO-Sar, —CH(COOH)₂, —N(CH₂COOH)₂, SO₃H, —PO₃H or absent; m, n, and p are each an integer from 1 to 25.

In the present invention, the structure of general formula 3 is

wherein J and O_(L) are defined as the above and R₁ is —OH or —NH₂;

In the present invention, the structure of general formula 4 is

wherein R₁ and O_(L) are defined as the above.

In one embodiment, the sequence of the GLP-1 receptor binding polypeptide is HX_(G2)EGTFTSDX_(G10)SSYLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)GRX_(G31) (SEQ ID NO:232), in which each of the variables is defined as the above.

In one embodiment, the sequence of the GLP-1 receptor binding polypeptide on the basis of exendin-4 is

-   X_(E1)X_(E2)X_(E3)GTX_(E6)TSDX_(E10)SX_(E12)X_(E13)X_(E14)EX_(E16)X_(E17)AX_(E19)X_(E20)X_(E21)FX_(E23)X_(E24)X_(E25)LX_(E27)X_(E28)     X_(E29)X_(E30)X_(E31)X_(E32)X_(E33)X_(E34)X_(E35)X_(E36)X_(E37)X_(E38)X_(E39)     (SEQ ID NO:233), wherein -   X_(E1) is a histidine, D-histidine, deamino-histidine,     β-hydroxyl-histidine, homohistidine, α-fluoromethyl-histidine,     α-methyl-histidine, N-methyl-histidine, N^(α)-acetyl-histidine,     α-methyl-histidine, 2-pyridinyl-alanine, 3-pyridinyl-alanine,     4-pyridinyl-alanine, imidazopropionyl, arginine, or tyrosine; X_(E2)     is a glycine, alanine, 2-methylalanine, D-serine, serine, threonine,     leucine, isoleucine, valine, aminocyclopropanecarboxylic acid,     aminocyclobutanecarboxylic acid, aminocyclopentanec arboxylic acid,     aminocyclohexanecarboxylic acid, aminocycloheptanecarboxylic acid,     or aminocyclooctanecarboxylic acid; X_(E3) is an aspartic acid,     glutamic acid, or glutamine; X_(E6) is a phenylalanine, alanine,     tyrosine, or naphthylalanine; X_(E10) is a leucine, isoleucine,     tyrosine, valine, alanine, lysine, cysteine, general formula 1,     general formula 2, or pentaglycine; X_(E12) is a lysine, cysteine,     arginine, serine, isoleucine, or general formula 1, or general     formula 2; X_(E13) is a glutamine, alanine, or tyrosine; X_(E14) is     a methionine, leucine, norleucine, isoleucine, alanine, valine, or     general formula 1, or general formula 2; X_(E16) is a glutamic acid,     aspartic acid, serine, glycine, lysine, or arginine; X_(E17) is a     glutamic acid, glutamine, arginine, or isoleucine; X_(E19) is a     valine, alanine, or glutamine; X_(E20) is a lysine, glutamine,     cysteine, arginine, general formula 1, or general formula 2; X_(E21)     is a leucine, glutamic acid, or aspartic acid; X_(E23) is an     isoleucine, leucine, valine, pentaglycine; X_(E24) is an alanine,     glutamic acid, aspartic acid, asparagine, or glutamine; X_(E25) is     an alanine, tryptophan, phenylalanine, tyrosine, cysteine, lysine,     naphthylalanine, general formula 1, or general formula 2; X_(E27) is     a lysine, cysteine, asparagine, leucine, valine, arginine, general     formula 1, or general formula 2; X_(E28) is an asparagine, lysine,     cysteine, arginine, alanine, general formula 1, or general formula     2; X_(E29) is —NH₂, glycine, glutamine, threonine, lysine, cysteine,     or absent; X_(E30) is —NH₂, glycine, tyrosine, arginine, lysine,     cysteine, general formula 1, general formula 2, or absent; X_(E31)     is —NH₂, glycine, proline, homoproline, thioproline, N-alkyl     alanine, or absent; X_(E32) is a serine, lysine, cysteine, deletion,     general formula 1, or general formula 2; X_(E33) is a serine,     lysine, cysteine, general formula 1, general formula 2, or absent;     X_(E34) is a glycine, absent, general formula 1, or general formula     2; X_(E35) is an alanine, lysine, cysteine, general formula 1,     general formula 2, or absent; X_(E36) is a proline, homoproline,     thioproline, N-alkyl alanine, or absent; X_(E37) is a proline,     homoproline, thioproline, N-alkyl alanine, or absent; X_(E38) is a     proline, homoproline, thioproline, N-alkylalanine, or absent;     X_(E39) is a serine, serine-NH₂, cysteine, cysteine-NH₂, lysine,     lysine-NH₂, or absent, or is a linking or spacer group + X_(E40),     wherein X_(E40) is a lysine, cysteine, general formula 3, or general     formula 4.

In one embodiment, the sequence of the GLP-1 receptor binding polypeptide on the basis of exendin-4 is

X_(E1)GEGTFTSDLSX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)X_(E30)X_(E31)X_(E32)X_(E33)X_(E34)X_(E35) X_(E36)X_(E37)X_(E38)X_(E39) (SEQ ID NO:234), wherein each of the variables is defined as the above.

In one embodiment, compared with the wild-type sequence, the amino acid sequence of an exendin-4 analogue have one or more amino acid residues substituted with a cysteine or lysine residue, for example, the C-terminal, the arginine at position 20, the tryptophan at position 25, the glycine at position 30, the alanine at position 35, and the serine at position 39 or the residues at other sites can be substituted with a cysteine or lysine residue.

From the above description, it can be seen that the GLP-1 receptor binding polypeptide and the interleukin-1 receptor antagonistic protein are able to protect β cells in the pancreas islet by similar or distinct mechanisms, thereby treating diabetes. Accordingly, the fusion protein, dimeric protein, cross-linking protein formed by connecting the two polypeptides may attain a therapeutic effect more excellent than that with either of the polypeptides alone, due to the synergism of the two polypeptides. Furthermore, one of the organs in which the GLP-1 receptors are mainly distributed is the pancreatic islet. The GLP-1 receptor binding polypeptide can serve as a guider for the fusion protein and enrich the interleukin-1 receptor antagonistic protein within and around the pancreatic islet, more effectively exerting actions such as anti-inflammation, etc.

The GLP-1 receptor binding polypeptide is linked to the interleukin-1 receptor antagonistic protein in various linkages through an alternative linking group (or spacer group).

(1) GLP-1 receptor binding polypeptide—linking group or spacer group-the interleukin-1 receptor antagonistic protein fusion protein.

In one embodiment, the GLP-1 receptor binding polypeptide and the interleukin-1 receptor antagonistic protein form a single chain compound via a linking group, of which the structure is (from left to right corresponding to the amino acid sequence from N-terminus to C-terminus):

-   the GLP-1 receptor binding polypeptide13 linking group (or spacer     group)-interleukin-1 receptor antagonistic protein; or -   the interleukin-1 receptor antagonistic protein-linking group (or     spacer group)-the GLP-1 receptor binding polypeptide.

In one embodiment, the sequence of the fusion protein on the basis of exendin-4 and the interleukin-1 receptor antagonistic protein is

X_(E1)X_(E2)X_(E3)GTX_(E6)TSDX_(E10)SX_(E12)X_(E13)X_(E14)EX_(E16)X_(E17)AX_(E19)X_(E20)X_(E21)FX_(E23)X_(E24)X_(E25)LX_(E27)X_(E28) X_(E29)X_(E30)X_(E31)X_(E32)X_(E33)X_(E34)X_(E35)X_(E36)X_(E37)X_(E38)X_(E39)-L_(j)-X_(IL0)RPSGRKSSKMQAFRIWDVNQK TFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGD ETRLQLEAVX_(IL84)ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFLX_(IL122)TAM EADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:235), wherein each of the variables is defined as the above.

In one embodiment, the sequence of the fusion protein on the basis of exendin-4 and the interleukin-1 receptor antagonistic protein is

X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)X_(E30)X_(E31)X_(E32)X_(E33)X_(E34) X_(E35)X_(E36)X_(E37)X_(E38)X_(E39)-L_(j)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQG PNVNLEEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)V KS GDETRLQLEAVX_(IL84)ITDLS ENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFLX_(IL122)TAMEADQPVS LTNMPDEGV MVTKFYFQEDE (SEQ ID NO:236), wherein each of the variables is defined as the above.

In one embodiment, the sequence of the fusion protein on the basis of exendin-4 and the interleukin-1 receptor antagonistic protein is

HX_(E2)EGTFTS DX_(E1)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)X_(E30)(PX_(E32)X_(E33)GX_(E35)PP PX_(E39))_(t2)-L_(j)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKI DVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVX_(IL84)ITDLSENRKQDKRFAFIR SDSGPTTSFESAAX_(IL116)PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:237), wherein t2 is 0 or 1 and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein on the basis of exendin-4 and the interleukin-1 receptor antagonistic protein is

HX_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)X_(E30)(PX_(E32)X_(E33)GX_(E35)PP PX_(E39))_(t2)-(GGGGS)_(m)-X_(L)-(GGGGS)_(n)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLV AGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVX_(IL84)IT DLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFLCTAMEADQPVSLTNMPDEG VMVTKFYFQEDE (SEQ ID NO:238), wherein m and n are each 0, 1, 2, 3, 4, 5, or 6; X_(L) is a cysteine, lysine, absent, general formula 1 or general formula 2; t2 is 0 or 1; and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein is HGEGTFTSDLSX_(E12)QMEEEAVRLFIEWLX_(E27)NGGPSSGAPPPS-(GGGGS)_(m)-X_(L)-(GGGG S)_(n)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIE PHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTS FESAAX_(IL116)PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:239), in which

m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein is HGEGTFTSDLSX_(E12)QMEEEAVRLFIEWLX_(E27)NGGPSSGAPPPX_(E39)-(GGGGS)n-X_(IL0)RPS GRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGI HGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:240), in which n is 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein is HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-(GGGGS)_(n)-X_(IL0)RPSGRKSS KMQAPRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGK MCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAC(S—O_(L))PG WFLC TAMEADQPVSLTNMPDEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO:241), wherein

n is 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein is HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-(GGGGS)_(n)-X_(IL0)RPSGRKSS KMQAPRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGK MCLSCVKSGDETRLQLEAVC (S—O_(L)) ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLC TAME ADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO:242), in which

n is 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein on the basis of GLP-1 and the interleukin receptor antagonistic protein is X_(G1)X_(G2)X_(G3)GX_(G5)X_(G6)TSDX_(G10)SX_(G12)YLEX_(G16)X_(G17)X_(G18)AX_(G20)X_(G21)FIX_(G24)X_(G25)LX_(G27)X_(G28)X_(G29)X_(G30)X_(G31)-L_(j)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKI DVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVX_(IL84)ITDLSENRKQDKR FAFIRSDSGPTTSFESAAX_(IL116)PGWFLX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQ EDE (SEQ ID NO:243); in which each of the variables is defined as the above.

In one embodiment, the sequence of the fusion protein is HX_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVX_(G28) GRX_(G31)-L_(j)-X_(IL0)RPSGRKSSK MQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKM CLSCVKSGDETRLQLEAVX_(IL84)ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PG WFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:244), in which each of the variables is defined as the above.

In one embodiment, the sequence of the fusion protein is HX_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)GRX_(G31)-(GGGGS)_(m)-X_(L)-(GG GG)_(n)-X_(IL0)RPSGRKSSKMQAPRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVX_(IL84)ITDLSENRKQDKRFAFIRSDSG PTTSFESAAX_(IL16)PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:245); in which m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein is HX_(G2)EGTFTSDX_(G10)SSYLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)GRG-(GGGGS)_(m)-X_(L)-(GGGGS)_(n)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPH ALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFE SAAX_(IL116)PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:246), in which m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein is HX_(G2)EGTFTSDX_(G10)SSYLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)GRX_(G31)-(GGGGS)_(n)-X_(IL0)RPSGR KSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHG GKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)P GWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:247), in which n is 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein is HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVKGRG-(GG GGS)_(n)-X_(IL0)RPSGRKSSKMQA PRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSC VKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAC(S—O_(L))PGWFLCT AME ADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO:248), wherein n is 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein is HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVKGRG-(GGGGS)_(n)-X_(IL0)RPSGRKSSKMQA PRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSC VKSGDETRLQLEAVC(S—O_(L))ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCT AME ADQPVSLTNMPDEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO:249), wherein n is 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein is HX_(G2)EGTFTSDX_(G10)SSYLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)GRPSSGAPPPS-(GGGGS)_(m)-X_(L)-(GGGGS)_(n)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKI DVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSD SGPTTSFESAAX_(IL116)CPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:250), wherein m and n are each 0, 1, 2, 3, 4, 5, or 6; X_(L) is a cysteine, lysine or absent, or is general formula 1 or general formula 2; and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein is HX_(G2)EGTFTSDX_(G10)SSYLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)GRPSSGAPPPX_(G39)-(GGGGS)_(n)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPH ALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFE SAAX_(IL116)PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:251), in which n is 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein is HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVKGRPSSGAPPPS-(GGGGS)_(n)-X_(IL0)RPSGR KSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHG GKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAC(S—O_(L)) PGWF LCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO:252), wherein n is 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein is HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVKGRPSSGAPPPS-(GGGGS)_(n)-X_(IL0)RPSGR KSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHG GKMCLSCVKSGDETRLQLEAVC(S—O_(L))ITDLSENRKQDKRFAFIRSDSGPTTSFESAAS PGWF LCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO:253),wherein n is 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

In one embodiment, the GLP-1 receptor binding polypeptide, the interleukin-1 receptor antagonistic protein, and a biomacromolecule form a single chain compound, for which the structure is (from left to right corresponding to the amino acid sequence from N-terminus to C-terminus):

GLP-1 receptor binding polypeptide - linking group (or spacer group)-biomacromolecule-linking group (or spacer group)-interleukin-1 receptor antagonistic protein;

GLP-1 receptor binding polypeptide - linking group (or spacer group)-interleukin-1 receptor antagonistic protein-linking group (or spacer group)-biomacromolecule; in which the biomacromolecule may be an albumin or IgG Fc, etc.

In one embodiment, the sequence of the fusion protein comprising the human albumin is

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-(GGGGS)_(m)-DAHKSEVAHR FKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLH TLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVM CTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVT DLTKVHTECCHGDLLECADDRADLAKYICENQDS IS S KLKECCEKPLLE KS HCIAEVE NDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRL AKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNA LLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCV LHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKE RQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKL VAASQAALGL-(GGGGS)_(n)-X_(IL0)RPSGRKSSKMQAPRIWDVNQKTFYLRNNQLVAGYL QGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKS GDETRLQLEAVNITD LS ENRK QDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE (SEQ ID NO:254), in which m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein with the human albumin is HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-(GGGGS)_(m)-X_(IL0)RPSGRKSS KMQAPRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGK MCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLC TAMEADQPVSLTNMPDEGVMVTKFYFQEDE-(GGGGS)_(n)-DAHKSEVAHRFKDLGEE NFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKL CTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHD NEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMP ADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYET TLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYT KKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQ TALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQA ALGL (SEQ ID NO:255), in which m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein with the human albumin is HX_(G2)EGTFTSDVSS YLEX_(G16)QAAKEFIAWLVKGRG-(GGGGS)_(m)-DAHKSEVAHRFKDL GEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFG DKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTA FHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLD ELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLT KVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEND EMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAK TYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALL VRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLH EKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQI KKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAA SQAALGL-(GGGGS)_(n)-X_(IL0)RPSGRKSSKMQAPRIWDVNQKTFYLRNNQLVAGYLQGP NVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDK RFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:256), wherein m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein with the human albumin is HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVKGRG-(GGGGS)_(m)-X_(IL0)RPSGRKSSKMQA PRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSC VKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAME ADQPVSLTNMPDEGVMVTKFYFQEDE-(GGGGS)_(n)-DAHKSEVAHRFKDLGEENFKAL VLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVAT LRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFL KKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKA SSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCH GDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSL AADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCC AAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQ VSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRV TKCCTESLVNRRPCFS ALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVE LVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO:257), wherein m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein with the human albumin is HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVKGRHX_(G2)EGTFTSDVSSYLEX_(G16)QAAKE FIAWLVKGR-(GGGGS)_(m)-RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGP NVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDK RFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE-(GGGGS)_(n)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFA KTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHK DDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKA AFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVAR LSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKL KECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFL YEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLI KQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAK RMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC CKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO:258), wherein m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein with the human albumin is HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVKGRHX_(G2)EGTFTSDVSSYLEX_(G16)QAAKE FIAWLVKGR-(GGGGS)_(m)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDH VKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQE PERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAP ELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGE RAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYI CENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAE AKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEF KPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA LEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL-(GGGGS)_(n)-RPSGRKSSK MQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKM CLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCT AMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:259); wherein m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

In one embodiment, the sequence of the fusion protein with the human albumin is HGEGTFTSDLSKQMEEEAVRLFIEWLKNGG(PSSGAPPPS)_(t2)HGEGTFTSDLSKQMEEE AVRLFIEWLKNGG(PSSGAPPPS)_(t2)-(GGGGS)_(m)-RPSGRKSSKMQAPRIWDVNQKTFYL RNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEA VNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDE GVMVTKFYFQEDE-(GGGGS)_(n)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCP PEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHP YFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASL QKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRA DLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVC KNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYA KVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNR RPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO:260), wherein m and n are each 0, 1, 2, 3, 4, 5, or 6 and t2 is 0 or 1.

In one embodiment, the sequence of the fusion protein with the human albumin is HGEGTFTSDLSKQMEEEAVRLFIEWLKNGG(PSSGAPPPS)_(t2)HGEGTFTSDLSKQMEEE AVRLFIEWLKNGG(PSSGAPPPS)_(t2)-(GGGGS)_(m)-DAHKSEVAHRFKDLGEENFKALVLI AFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLR ETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKK YLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA KQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDL LECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAAD FVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTP TLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCC TESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKH KPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL-(GGGGS)_(n)-RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPH ALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFE SAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:261), wherein m and n are each 0, 1, 2, 3, 4, 5, or 6 and t2 is 0 or 1.

In one embodiment, the sequence of the fusion protein with IgG1 Fc is HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-(GGGGS)_(m)-X_(IL0)RPSGRKSS KMQAPRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGK MCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLC TAMEADQPVSLTNMPDEGVMVTKFYFQEDE-(GGGGS)_(n)-AEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:262), wherein m and n are each 0, 1, 2, 3, 4, 5, or 6 and X_(mo) is a methionine, cysteine, general formula 1, general formula 2 or absent.

In one embodiment, the sequence of the fusion protein with IgG1 Fc is HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-(GGGGS)_(m)-AEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK-(G GGGS)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDV VPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSG PTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:263), wherein m and n are each 0, 1, 2, 3, 4, 5, or 6 and X_(IL0) is a methionine, cysteine, general formula 1, general formula 2 or absent.

In one embodiment, the sequence of the fusion protein with IgG1 Fc is HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVKGRG-(GGGGS)_(m)-X_(IL0)RPSGRKSSKMQA FRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSC VKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAME ADQPVSLTNMPDEGVMVTKFYFQEDE-(GGGGS)_(n)-AEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:264), wherein m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are individually defined as the above.

In one embodiment, the sequence of the fusion protein with IgG1 Fc is HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVKGRG-(GGGGS)_(m)-AEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK-(GGGGS)_(n)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPH ALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFE SAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:265), wherein m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are individually defined as the above.

The dimeric protein is a homodimer consisting of two identical sequences mentioned above, and connected by an interchain disulfide bond formed between the cysteines of the Fc portions of the two monomeric proteins.

In one embodiment, the sequence of the monomer in the fusion protein consisting of the IgG 4 Fc and the interleukin-1 receptor antagonistic protein is: HX_(G2)EGTFTSDVSSYLEEQAAKEFIAWLYKGGG-(GGGGS)_(m)-RPSGRKSSKMQAPRIW DVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSG DETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFLCTAMEAD QPYSLTNMPDEGYMYTKFYFQEDE-(GGGGS)_(n)-AESKYGPPCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEYTCNYYDVSQEDPEVQFNWYYDGVEYHNAKTKPREEQFNS TYRYYSYLTYLHQDWLNGKEYKCJCVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTC_(b)LYKGFYPSDIAVEWESNGQPENNYKTTPPYLDSDGSFFLYSRLTY DKSRWQEGNVFSC_(b)SVMHEALHNHYTQKSLSLSLG (SEQ ID NO:266), wherein m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

The dimeric protein is a homodimer consisting of two identical sequences mentioned above. Each cysteine C underlined and the cysteine at the corresponding position in the other monomer form an interchain disulfide bond; an intrachain disulfide bond is formed between the C_(a)s in each monomer; and an intrachain disulfide bond is formed between the C_(b)s in each monomer.

In one embodiment, the sequence of the monomer in the fusion protein consisting of the IgG 4 Fc and the interleukin-1 receptor antagonistic protein is: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGG-(PSSGAPPPS)_(t2)-(GGGGS)_(m)-RPSGRKSS KMQAPRIWDYNQKTFYLRNNQLVAGYLQGPNYNLEEKIDYYPIEPHALFLGIHGGK MCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGW FLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE-(GGGGS)_(n)-AESKYGPPCPPCPAPE AAGGPSVFLFPPKPKDTLMISRTPEYTC_(a)VVVDVSQEDPEVQFNWYYDGVEYHNAKT KPREEQFNSTYRYYSYLTYLHQDWLNGKEYKC_(a)KVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTC_(b)LYKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSC_(b)SVMHEALHNHYTQKSLSLSLG (SEQ ID NO:267), wherein m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as the above.

The dimeric protein is a homodimer consisting of two identical sequences mentioned above. Each cysteine C underlined and the cysteine at the corresponding position in the other monomer form an interchain disulfide bond; an intrachain disulfide bond is formed between the C_(a)s in each monomer; and an intrachain disulfide bond is formed between the C_(b)s in each monomer.

(2) the fusion protein consisting of the interleukin-1 receptor antagonistic protein—the linking group or spacer group—the GLP-1 receptor binding polypeptide

The interleukin-1 receptor antagonistic protein can be cross-linked to the GLP-1 receptor binding polypeptide through a linking group (or a spacer group), by using a cysteine at position 116 or introducing a cysteine at position 0 (or at the N-terminus), 6, 8, 9, 84, 141, or 153 (or at the C-terminus).

In one embodiment, the sequence of the fusion protein is

(SEQ ID NO: 268) U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNL EEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSEN RKQDKRFAFIRSDSGPTTSFESAAC (linking group-GLP-1 receptor bindingpolypeptide)PGWFLCTAMEADQPVSLTNMP DEGVMVTKFYFQEDE;

In another embodiment, the sequence of the fusion protein is

(SEQ ID NO: 269) U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNL EEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC (linking group-GLP-1 receptor binding polypeptide) ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVS LTNMPDEGVMVTKFYFQEDE;

wherein U_(L) is a structure of -W-X-Y-Z, fatty acid, polyethylene glycol, albumin, IgG Fc, glycosyl, hydrogen atom, N^(α)—(N^(α)—(HOOC(CH₂)_(n)CO)-γ-Glu)-, or N^(α)—(N^(α)—(CH₃(CH₂)_(n)CO)-γ-Glu)-, in which n is an integer from 8 to 20, such as 10, 12, 14, 16, 18 or 20; N^(α)indicates the α-amino group of an amino acid or amino acid residue or general formula 5; and other variables are individually defined as the above. The structure of general formula 5 is

In one particular embodiment, the molecular structure of the linking group may be maleimide-PEG-maleimide or I—CH₂—CONH-PEG-NHCO—CH₂—I; in one particular embodiment, the molecular structure of the linking group may be maleimide-(CH₂)_(n)-maleimide or I—CH₂—CONH-(CH₂)_(n)—NHCO—CH₂—I, in which n may be an integer from 1 to 30; in one particular embodiment, the molecular structure of the linking group may be maleimide-PEG-NHS. The GLP-1 receptor binding polypeptide may typically react with the linking group through the amino or mercapto group on the side chain of an amino acid residue.

In one embodiment, the sequence of the GLP-1 receptor binding polypeptide may be

(SEQ ID NO: 270) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)X_(E30)(PX_(E32)X_(E33)G X_(E35)PPPC)_(t2); (SEQ ID NO: 271) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)X_(E30)(PX_(E32)X_(E33)G CPPPX_(E39))_(t2); (SEQ ID NO: 272) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)X_(E30)(PX_(E32)CGX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 273) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)X_(E30)(PCX_(E33)GX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 274) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)C(PX_(E32)X_(E33)GX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 275) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)CX_(E30)(PX_(E32)X_(E33)GX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 276) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)CX_(E29)X_(E30)(PX_(E32)X_(E33)GX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 277) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLCX_(E28)X_(E29)X_(E30)(PX_(E32)X_(E33)GX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 278) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVCLFIEWIA_(E27)X_(E28)X_(E29)X_(E30)(PX_(E32)X_(E33)GX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 279) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QCEEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)X_(E30)(PX_(E32)X_(E33)GX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 280) X_(E1)X_(E2)EGTFTSDX_(E10)SCQX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)X_(E30)(PX_(E32)X_(E33)GX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 281) X_(E1)X_(E2)EGTFTSDCSX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)X_(E30)(PX_(E32)X_(E33)GX_(E35) PPPX_(E39))_(t2); the variables in the individual sequences are individually defined as the above.

In one particular embodiment, the sequence of the GLP-1 receptor binding polypeptide may be one of the following sequences:

(SEQ ID NO: 282) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC; (SEQ ID NO: 185) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPC; (SEQ ID NO: 284) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGCPPPS; (SEQ ID NO: 285) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSCGAPPPS; (SEQ ID NO: 286) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPCSGAPPPS; (SEQ ID NO: 287) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGCPSSGAPPPS; (SEQ ID NO: 288) HGEGTFTSDLSKQMEEEAVRLFIEWLKNCGPSSGAPPPS; (SEQ ID NO: 289) HGEGTFTSDLSKQMEEEAVRLFIEWLKCGGPSSGAPPPS; (SEQ ID NO: 188) HGEGTFTSDLSKQMEEEAVRLFIEWLCNGGPSSGAPPPS; (SEQ ID NO: 291) HGEGTFTSDLSKQMEEEAVRLFIECLKNGGPSSGAPPPS; (SEQ ID NO: 292) HGEGTFTSDLSKQMEEEAVCLFIEWLKNGGPSSGAPPPS; (SEQ ID NO: 293) HGEGTFTSDLSKQCEEEAVRLFIEWLKNGGPSSGAPPPS; (SEQ ID NO: 294) HGEGTFTSDLSCQMEEEAVRLFIEWLKNGGPSSGAPPPS; (SEQ ID NO: 295) HGEGTFTSDCSKQMEEEAVRLFIEWLKNGGPSSGAPPPS; (SEQ ID NO: 296) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGC.

The GLP-1 receptor binding polypeptide is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a cysteine with the maleimide or iodoacetyl on the linking group.

In one embodiment, the sequence of the GLP-1 receptor binding polypeptide may be

(SEQ ID NO: 297) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)X_(E30)(PX_(E32)X_(E33)G X_(E35)PPPK)_(t2); (SEQ ID NO: 298) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)X_(E30)(PX_(E32)X_(E33)G KPPPX_(E39))_(t2); (SEQ ID NO: 299) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)X_(E30)(PX_(E32)KGX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 300) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)X_(E30)(PKX_(E33)GX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 301) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)K(PX_(E32)X_(E33)GX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 302) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)KX_(E30)(PX_(E32)X_(E33)GX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 303) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)KX_(E29)X_(E30)(PX_(E32)X_(E33)GX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 304) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLKX_(E28)X_(E29)X_(E30)(PX_(E32)X_(E33)GX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 305) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QX_(E14)EEEAVKLFIEWLX_(E27)X_(E28)X_(E29)X_(E30)(PX_(E32)X_(E33)GX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 306) X_(E1)X_(E2)EGTFTSDX_(E10)SX_(E12)QKEEEAVX_(E20)LFIEWLX_(E27)X_(E28)XENX_(E30)(PX_(E32)X_(E33)GX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 307) X_(E1)X_(E2)EGTFTSDX_(E10)SKQX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)X_(E30)(PX_(E32)X_(E33)GX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 308) X_(E1)X_(E2)EGTFTSDKSX_(E12)QX_(E14)EEEAVX_(E20)LFIEWLX_(E27)X_(E28)X_(E29)X_(E30)(PX_(E32)X_(E33)GX_(E35) PPPX_(E39))_(t2);

the variables in the individual sequences are individually defined as the above.

In one particular embodiment, the sequence of the GLP-1 receptor binding polypeptide may be one of the following sequences:

(SEQ ID NO: 309) HGEGTFTSDLSRQMEEEAVRLFIEWLRNGGPSSGAPPPSK; (SEQ ID NO: 310) HGEGTFTSDLSRQMEEEAVRLFIEWLRNGGPSSGAPPPK; (SEQ ID NO: 311) HGEGTFTSDLSRQMEEEAVRLFIEWLRNGGPSSGKPPPS; (SEQ ID NO: 312) HGEGTFTSDLSRQMEEEAVRLFIEWLRNGGPSKGAPPPS; (SEQ ID NO: 313) HGEGTFTSDLSRQMEEEAVRLFIEWLRNGGPKSGAPPPS; (SEQ ID NO: 314) HGEGTFTSDLSRQMEEEAVRLFIEWLRNGKPSSGAPPPS; (SEQ ID NO: 315) HGEGTFTSDLSRQMEEEAVRLFIEWLRNKGPSSGAPPPS; (SEQ ID NO: 316) HGEGTFTSDLSRQMEEEAVRLFIEWLRKGGPSSGAPPPS; (SEQ ID NO: 317) HGEGTFTSDLSRQMEEEAVRLFIEWLKNGGPSSGAPPPS; (SEQ ID NO: 318) HGEGTFTSDLSRQMEEEAVRLFIEKLRNGGPSSGAPPPS; (SEQ ID NO: 319) HGEGTFTSDLSRQMEEEAVKLFIEWLRNGGPSSGAPPPS; (SEQ ID NO: 320) HGEGTFTSDLSRQKEEEAVRLFIEWLRNGGPSSGAPPPS; (SEQ ID NO: 321) HGEGTFTSDLSKQMEEEAVRLFIEWLRNGGPSSGAPPPS; (SEQ ID NO: 322) HGEGTFTSDKSRQMEEEAVRLFIEWLRNGGPSSGAPPPS; (SEQ ID NO: 323) HGEGTFTSDLSRQMEEEAVRLFIEWLRNGK-NH₂.

The GLP-1 receptor binding polypeptide is reacted with the N-hydroxyl succinimidyl ester of the linking group through the amino group on the side chain of a lysine, thus linking to the interleukin-1 receptor antagonistic protein.

In one embodiment, the sequence of the GLP-1 receptor binding polypeptide may be

(SEQ ID NO: 324) X_(G1)X_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)X_(G29)X_(G30)(PX_(E32)X_(E33)GX_(E35) PPPC)_(t2); (SEQ ID NO: 325) X_(G1)X_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)X_(G29)X_(G30)(PX_(E32)X_(E33)GC PPPX_(E39))_(t2); (SEQ ID NO: 326) X_(G1)X_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)X_(G29)X_(G30)(PX_(E32)CGX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 327) X_(G1)X_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)X_(G29)X_(G30)(PCX_(E33)GX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 328) X_(G1)X_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)X_(G29)X_(G30)C; (SEQ ID NO: 329) X_(G1)X_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)X_(G29)C-NH₂; (SEQ ID NO: 330) X_(G1)X_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)CX_(G30)X_(G31); (SEQ ID NO: 331) X_(G1)X_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVCX_(G29)X_(G30)X_(G31); (SEQ ID NO: 332) X_(G1)X_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAX_(G20)EFIACLVX_(G28)X_(G29)X_(G30)X_(G31); (SEQ ID NO: 333) X_(G1)X_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAACEFIAWLVX_(G28) X_(G29)X_(G30)X_(G31); (SEQ ID NO: 334) X_(G1)X_(G2)EGTFTSDX_(G10)SCYLEX_(G16)QAAX_(G20)EFIAWLVX_(G28) X_(G29)X_(G30)X_(G31); (SEQ ID NO: 335) X_(G1)X_(G2)EGTFTSDCSX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVX_(G28) X_(G29)X_(G30)X_(G31); the variables in the individual sequences are individually defined as the above.

In one particular embodiment, the sequence of the GLP-1 receptor binding polypeptide may be one of the following sequences:

(SEQ ID NO: 336) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVKGRPSSGAPPPC; (SEQ ID NO: 337) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVKGRPSSGCPPPS; (SEQ ID NO: 338) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVKGRPSCGAPPPS; (SEQ ID NO: 339) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVKGRPCSGAPPPS; (SEQ ID NO: 340) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVKGRC; (SEQ ID NO: 341) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVKGC-NH₂; (SEQ ID NO: 342) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVKCRG; (SEQ ID NO: 343) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVCGRG; (SEQ ID NO: 344) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVCGR-NH₂; (SEQ ID NO: 345) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIACLVKGRG; (SEQ ID NO: 346) HX_(G2)EGTFTSDVSSYLEX_(G16)QAACEFIAWLVKGRG; (SEQ ID NO: 347) HX_(G2)EGTFTSDVSSYLEX_(G16)QAACEFIAWLVKGR-NH₂; (SEQ ID NO: 348) HX_(G2)EGTFTSDVSCYLEX_(G16)QAAKEFIAWLVKGRG; (SEQ ID NO: 349) HX_(G2)EGTFTSDCSSYLEX_(G16)QAAKEFIAWLVKGRG; (SEQ ID NO: 350) HX_(G2)EGTFTSDCSSYLEX_(G16)QAAKEFIAWLVKGR-NH₂; the variables in the individual sequences are individually defined as the above.

The polypeptide is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a cysteine with the maleimide or iodoacetyl on one terminus of the linking group.

In one embodiment, the sequence of the GLP-1 receptor binding polypeptide may be:

(SEQ ID NO: 351) X_(G1)X_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)X_(G29)X_(G30)(PX_(E32)X_(E33)GX_(E35) PPPK)_(t2); (SEQ ID NO: 352) X_(G1)X_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)X_(G29)X_(G30)(PX_(E32)X_(E33)GK PPPX_(E39))_(t2); (SEQ ID NO: 353) X_(G1)X_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)X_(G29)X_(G30)(PX_(E32)KGX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 354) X_(G1)X_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)X_(G29)X_(G30)(PKX_(E33)GX_(E35) PPPX_(E39))_(t2); (SEQ ID NO: 355) X_(G1)X_(G2)EGTFTSDX_(G11)3SX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)X_(G29)X_(G30)K; (SEQ ID NO: 356) X_(G1)X_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)X_(G29)K-NH₂; (SEQ ID NO: 357) X_(G1)X_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)10CG3DX_(G31); (SEQ ID NO: 358) X_(G1)X_(G2)EGTFTSDX_(G11)3SX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVKX_(G29)X_(G30)X_(G31); (SEQ ID NO: 359) X_(G1)X_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAX_(G20)EFIAKLVX_(G28)X_(G29)X_(G30)X_(G31); (SEQ ID NO: 360) X_(G1)X_(G2)EGTFTSDX_(G10)SX_(G12)YLEX_(G16)QAAKEFIAWLVX_(G28)X_(G29)X_(G30)X_(G31); (SEQ ID NO: 361) X_(G1)X_(G2)EGTFTSDX_(G10)SKYLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)X_(G29)X_(G30)X_(G31); (SEQ ID NO: 362) X_(G1)X_(G2)EGTFTSDKSX_(G12)YLEX_(G16)QAAX_(G20)EFIAWLVX_(G28)X_(G29)X_(G30)X_(G31); and the variables in the individual sequences are individually defined as the above.

In one particular embodiment, the sequence of the GLP-1 receptor binding polypeptide may be one of the following sequences:

(SEQ ID NO: 363) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAREFIAWLVRGRPSSGAPPPK; (SEQ ID NO: 364) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAREFIAWLVRGRPSSGKPPPS; (SEQ ID NO: 365) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAREFIAWLVRGRPSKGAPPPS; (SEQ ID NO: 366) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAREFIAWLVRGRPKSGAPPPS; (SEQ ID NO: 367) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAREFIAWLVRGRK; (SEQ ID NO: 368) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAREFIAWLVRGK-NH₂; (SEQ ID NO: 369) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAREFIAWLVRKRG; (SEQ ID NO: 370) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAREFIAWLVKGRG; (SEQ ID NO: 371) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAREFIAKLVRGRG; (SEQ ID NO: 372) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVRGRG; (SEQ ID NO: 373) HX_(G2)EGTFTSDVSSYLEX_(G16)QAAKEFIAWLVRGR-NH₂; (SEQ ID NO: 374) HX_(G2)EGTFTSDVSKYLEX_(G16)QAAREFIAWLVRGRG; (SEQ ID NO: 375) HX_(G2)EGTFTSDKSSYLEX_(G16)QAAREFIAWLVRGRG; and (SEQ ID NO: 376) HX_(G2)EGTFTSDKSSYLEX_(G16)QAAREFIAWLVRGR-NH₂; the variables in the individual sequences are individually defined as the above.

The GLP-1 receptor binding polypeptide is reacted with the N-hydroxyl succinimidyl ester of the linking group through the amino group on the side chain of a lysine, thus linking to the interleukin-1 receptor antagonistic protein.

In one embodiment, the sequence of the fusion protein is

(GLP-1 receptor binding polypeptide linking group)-C_(IL0)—X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKI DVVPIEPHALFLGIH GGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVX_(IL84)ITDLSENRKQDKRF AFIRSDSGPTTSFESAAX_(IL116)PGWFLX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQE DE (SEQ ID NO:377); or

C_(IL0)(linking group—GLP-1 receptor binding polypeptide) X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNN-QLVAGYLQGPNVNLEEKIDVVPIEP HALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSF ESAAX_(IL116)PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:378); or

U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKI DVVPIEPHALFLGIH GGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVX_(Il84)ITDLSENRKQDKR FAFIRSDSGPTTSFESAAX_(IL116)PGWFLX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQ EDEC_(IL153) (linking group—GLP-1 receptor binding polypeptide) (SEQ ID NO:379); wherein

C_(IL0) and C_(IL153) are cysteines or absent and other variables are respectively defined as the above. The linking group is defined as herein. In one embodiment, the linking group has an aldehyde group and is linked to the N-terminus of the interleukin-1 receptor antagonistic protein through reductive amination; in another embodiment, C_(IL0) cysteine is linked to the linking group via the mercapto group on the side chain, and then is linked to the GLP-1 receptor binding polypeptide.

The GLP-1 receptor binding polypeptide may typically react with the linking group through the amino or mercapto group on the side chain of an amino acid residue.

In one embodiment, the GLP-1 receptor binding polypeptide is a modified conjugate. The sequences of the fusion protein /dimeric protein/cross-linking protein of the GLP-1 receptor binding polypeptide and the interleukin-1 receptor antagonistic protein are selected from:

G-1:

G-1: (SEQ ID NO: 1) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSRPSGRKSSKMQAF RIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCV KSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEA DQPVSLTNMPDEGVMVTKFYFQEDE; G-2: (SEQ ID NO: 2) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSRPSGRKSS KMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGK MCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLC TAMEADQPVSLTNMPDEGVMVTKFYFQEDE; G-3: (SEQ ID NO: 3) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSRPS GRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGI HGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPG WFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE; G-4: (SEQ ID NO: 4) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSGG GGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEP HALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSF ESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE; G-5: (SEQ ID NO: 5) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGRPSGRKSSKMQAFRIWDVNQK TFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRL QLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTN MPDEGVMVTKFYFQEDE; G-6: (SEQ ID NO: 6) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGGGSRPSGRKSSKMQAFRIWDV NQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDE TRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSL TNMPDEGVMVTKFYFQEDE; G-7: (SEQ ID NO: 7) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGGGSGGGGSRPSGRKSSKMQAF RIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCV KSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEA DQPVSLTNMPDEGVMVTKFYFQEDE; G-8: (SEQ ID NO: 8) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGGGSGGGGSGGGGSRPSGRKSS KMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGK MCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLC TAMEADQPVSLTNMPDEGVMVTKFYFQEDE; G-9: (SEQ ID NO: 9) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSRPSGRKSS KMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGK MCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAC[ S -maleimide- (CH₂)4—NH—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-Glu)]PGWFLCTAMEADQPVSLTNMPDEGVM VTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-10: (SEQ ID NO: 10) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSRPSGRKSS KMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGK MCLSCVKSGDETRLQLEAVC( S —CH₂—CONH-PEG20K)ITDLSENRKQDKRFAFIRSDSG PTTSFESAASPG WFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-11: (SEQ ID NO: 11) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGGGGSGGGGSRPSGRKSSKMQA FRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSC VKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAC( S —CH₂—CONH-PEG20K) PGWF LCTA MEADQPVSLTNMPDEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue); G-12: (SEQ ID NO: 12) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSRPS GRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGI HGGKMCLSCVKSGDETRLQLEAVC[ S -maleimide-(CH₂)₄—NH—(N^(α)—(HOOC(CH₂)₁₄CO)-γ- Glu)]ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPD EGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-13: (SEQ ID NO: 13) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGGGSRPSGRKSSKMQAFRIWDV NQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDE TRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAC( S -maleimide-PEG20K)PG WFLCTAMEAD QPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-14: (SEQ ID NO: 14) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGGGSRPSGRKSSKMQAFRIWDV NQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDE TRLQLEAVC[ S -maleimide-(CH₂)₄—NH—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-Glu)]ITDLSENRKQDK RFAFIRS DSGPTTSFESAASPGWFLCTAME ADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-15: (SEQ ID NO: 15) HGEGTFTSDLSKQMEEEAVRLFIEWLC[ S -maleimide-(CH₂)₄—NH—(N^(α)—(HOOC(CH₂)₁₄CO)- γ-Glu)]NGGPSSGAPPPSGGGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQ LVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNIT DLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMV TKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-16: (SEQ ID NO: 16) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPC( S —CH₂—CONH-PEG20K) GGGG SGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPT TSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-17: (SEQ ID NO: 17) HGEGTFTSDLSKQMEEEAVC[ S -maleimide-(CH₂)₄—NH—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-Glu)] LFIEWLKNGGPSSGAPPPSGGGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNN QLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNI TDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVM VTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-18: (SEQ ID NO: 18 HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGRPSGRKSSKMQAFRIWDVNQ KTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETR LQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTN MPDEGVMVTKFYFQEDE; G-19: (SEQ ID NO: 19 HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGSRPSGRKSSKMQAFRIW DVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSG DETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQP VSLTNMPDEGVMVTKFYFQEDE; G-20: (SEQ ID NO: 20 HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGSGGGGSRPSGRKSSKMQ AFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLS CVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAME ADQPVSLTNMPDEGVMVTKFYFQEDE; G-21: (SEQ ID NO: 21 HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGSGGGGSGGGGSRPSGRK SSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGG KMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFL CTAMEADQPVSLTNMPDEGVMVTKFYFQEDE; G-22: (SEQ ID NO: 22) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRPSSGAPPPSGGGGSGGGGSRPS GRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGI HGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPG WFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE; G-23: (SEQ ID NO: 23) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGRPSGRKSSKMQAFRIWDVNQ KTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETR LQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAC( S —CH₂—CONH-PEG20K)PGW FLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-24: (SEQ ID NO: 24) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGSRPSGRKSSKMQAFRIW DVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSG DETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAC( S —CH₂—CONH-PEG20K) PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue); G-25: (SEQ ID NO: 25) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGSRPSGRKSSKMQAFRIW DVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSG DETRLQLEAVC[ S -maleimide-(CH₂)₄—NH—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-Glu)]ITDLSENRKQ DKRFAFIRS DSGPTTSFESAASPGWFLCTAME ADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-26: (SEQ ID NO: 26) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGSGGGGSRPSGRKSSKMQ AFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLS CVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAC( S —CH₂—CONH- PEG20K)PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-27: (SEQ ID NO: 27) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGSGGGGSRPSGRKSSKMQ AFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLS CVKSGDETRLQLEAVC[ S -maleimide-(CH₂)₄—NH—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-Glu)]ITDLS ENRKQDKR FAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-28: (SEQ ID NO: 28) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGSGGGGSGGGGSRPSGRK SSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGG KMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAC( S —CH₂—CONH- PEG20K)PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-29: (SEQ ID NO: 29) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRPSSGAPPPSGGGGSGGGGSRPS GRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGI HGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAC[ S - maleimide-(CH₂)₄—NH—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-Glu)]PGWFLCTAMEADQPVSLTNMPD EGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-30: (SEQ ID NO: 30) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPC( S -maleimide-(CH₂)₁₅—COOH) GGGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNV NLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRF AFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-31: (SEQ ID NO: 31) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPC( S -maleimide-(CH₂)₁₇—COOH) GGGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNV NLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRF AFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-32: (SEQ ID NO: 32) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPC [ S —CH₂—CONH—(CH₂CH₂O)₂—(CH₂)₂NH—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-Glu)]- GGGGSGGGGSRPSGRKSSKMQAF RIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCV KSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEAD QPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-33: (SEQ ID NO: 33) HGEGTFTSDLSKQMEEEAVRLFIEWLC [ S —CH₂—CONH—(CH₂CH₂O)₂—(CH₂)₂—NH—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-Glu)] NGGPSSGAPPPSGGGGSGGGGSRPSGRKSSKMQAFRI WDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVK SGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQ PVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-34: (SEQ ID NO: 34) HGEGTFTSDLSKQMEEEAVC [ S —CH₂—CONH—(CH₂CH₂O)₂—(CH₂)₂—NH—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-Glu)] LFIEWLKNGGPSSGAPPPSGGGGSGGGGSRPSGRKSSKMQAFR IWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVK SGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQ PVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-35: (SEQ ID NO: 35) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPC [ S —CH₂—CONH—(CH₂CH₂O)₂—(CH₂)₂—NH—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-Glu)] SGAPPPSGGGGSGGGGSRPSGRKSSKMQAFRI WDVNQKTF YLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQL EAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMP DEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-36: (SEQ ID NO: 36) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSRPS GRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGI HGGKMCLSCVKSGDETRLQLEAVC( S -maleimide—(CH₂)₁₅—COOH)ITDLSENRKQDKRF AFTRSDSGPT TSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-37: (SEQ ID NO: 37) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSRPS GRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGI HGGKMCLSCVKSGDETRLQLEAVC[ S —CH₂CONH—(CH₂CH₂O)₂—(CH₂)₂—NH—(N^(α)—(HOOC(CH₂)₁₄CO)- γ-Glu)]ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQ PVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-38: (SEQ ID NO: 38) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSRPS GRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGI HGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAC( S - maleimide-(CH₂)₁₇—COOH)-PGWFLCTAMEADQPVSLTNMPDEGVMVTKEYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-39: (SEQ ID NO: 39) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSRPS GRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGI HGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAC[ S - CH₂—CONH—(CH₂CH₂O)₄—(CH₂)₂—NH—(N^(α)—(HOOC(CH₂)₁₆CO)-γ-Glu)]-PGWFLCTAMEAD QPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-40: (SEQ ID NO: 40) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRC( S -maleimide-(CH₂)₁₅—COOH)GG GGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDV VPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSG PTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-41: (SEQ ID NO: 41) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRC [ S —CH₂—CONH—(CH₂CH₂O)₂—(CH₂)₂—NH—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-Glu)] GGGGSGGGGSRPSGRKSSKMQAFRIWDVNQK TFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRL QLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTN MPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-42: (SEQ ID NO: 42) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGSGGGGSRPSGRKSSKMQ AFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLS CVKSGDETRLQLEAVC( S -maleimide-(CH₂)₁₇—COOH)ITDLSENRKQDKRFAFIRSDSGP TTSFESAASPG WFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-43: (SEQ ID NO: 43) HGEGTFTSDVSSYLEEQAAC[ S —CH₂—CONH—(CH₂CH₂O)₂—(CH₂)₂—NH—(N^(α)—(HOOC(CH₂)₁₄CO)- γ-Glu)]EFIAWLVKGRGGGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTF YLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQL EAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMP DEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-44: (SEQ ID NO: 44) HAEGTFTSDVSSYLEGQAAC[ S —CH₂—CONH—(CH₂CH₂O)₂—(CH₂)₂—NH—(N^(α)—(HOOC(CH₂)₁₄CO)- γ-Glu)]EFIAWLVKGRGGGGSGGGGSRPSGRKSSKMQAFRIWDVNQK TFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRL QLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTN MPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-45: (SEQ ID NO: 45) HGEGTFTSDC[ S -CH₂—CONH—(CH₂CH₂O)₂—(CH₂)₂—NH—(N^(α)—(HOOC(CH₂)₁₄CO-γ- Glu-N-γ-Glu)]SSYLEEQAAKEFIAWLVKGRGGGGSGGGGSRPSGRKSSKMQAFRIWD VNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGD ETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVS LTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-46: (SEQ ID NO: 46) HAEGTFTSDC[ S —CH₂—CONH—(CH₂CH₂O)₂—(CH₂)₂—NH—(N^(α)—(HOOC(CH₂)₁₄CO)-γ- Glu)]SSYLEGQAAKEFIAWLVKGRGGGGSGGGGSRPSGRKSSKMQAFRIWDVNQKT FYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQ LEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMP DEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-47: (SEQ ID NO: 47) HGEGTFTSDVSSYLEEQAAKEFIAWLVC [ S —CH₂—CONH—(CH₂CH₂O )₂—(CH₂)₂—NH—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-Glu)] GRGGGGSGGGGSRPSGRKSSKMQAFRIWDVNQKT FYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQ LEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMP DEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); G-48: (SEQ ID NO: 48) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGSGGGGSRPSGRKSSKMQ AFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLS CVKSGDETRLQLEAVC[ S —CH₂—CONH—(CH₂CH₂O)₄—(CH₂)₂—NH—(N^(α)—(HOOC(CH₂)₁₆CO)- γ-Glu)]ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMP DEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue); G-49: (SEQ ID NO: 49) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGSGGGGSRPSGRKSSKMQ AFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLS CVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAC( S -maleimide-(CH₂)₁₅—COOH) PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue); G-50: (SEQ ID NO: 50) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGSGGGGSRPSGRKSSKMQ AFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLS CVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAC [ S —CH₂—CONH—(CH₂CH₂O)₂—(CH₂)₂—NH—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-Glu)] PGWFLCTAMEADQPVSLTNMP DEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue); G-51:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC (S-maleimide-PEG11-maleimide-S-GLP-1 receptor binding polypeptide) ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDE GVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:51), wherein the GLP-1 receptor binding polypeptide has the following sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPC (SEQ ID NO:185) and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a terminal cysteine residue with the maleimide;

G-52:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTT SFESAAC (S-maleimide-PEG11-maleimide-S-GLP-1 receptor binding polypeptide) PGWFLCTAMEADQPVSLT NMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:52), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO: 185 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a terminal cysteine residue with the maleimide;

G-53:

RPSGRKSS KMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC(S-maleimide-PEG11-maleimide-S-GL P-1 receptor binding polypeptide)ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLT NMPDE GVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO:53), wherein the GLP-1 receptor binding polypeptide has the following sequence: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRC(SEQ ID NO:186) and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a terminal cysteine residue with the maleimide;

G-54:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTT SFE SAAC (S-maleimide-PEG11-maleimide-S-GLP-1 receptor binding polypeptide) PGWFLCTAMEADQPVSLT NMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:54), wherein the the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO: 186 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a terminal cysteine residue with the maleimide;

G-55:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC(S-maleimide-PEG11-maleimide-S-GL P-1 receptor binding polypeptide)ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLT NMPDE GVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO:55), wherein the GLP-1 receptor binding polypeptide has the following sequence: HGEGTFTSDVSSYLEEQAAKEFIAWLVCGRG (SEQ ID NO:187) and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a cysteine residue with the maleimide;

G-56:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTT SFESAAC (S-maleimide-PEG11-maleimide-S-GLP-1 receptor binding polypeptide) PGWFLCTAMEADQPVSLTNM PDEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO: 56), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO:187 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a cysteine residue with the maleimide;

G-57:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC(S-maleimide-PEG11-maleimide-S-GL P-1 receptor binding polypeptide)ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLT NMPDE GVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO:57), wherein the GLP-1 receptor binding polypeptide has the following sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLCNGGPSSGAPPPS (SEQ ID NO:188) and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a cysteine residue with the maleimide;

G-58:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTT SFESAAC (S-maleimide-PEG11-maleimide-S-GLP-1 receptor binding polypeptide) PGWFLCTAMEADQPVSLTN-MPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:58), wherein the the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO: 188 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a cysteine residue with the maleimide;

G-59:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC (S-maleimide-PEG20K-maleimide-S-GLP-1 receptor binding polypeptide) ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTA MEADQPVSLTNMPDE GVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:59), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO: 185 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a terminal cysteine residue with the maleimide;

G-60:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTT SFESAAC (S-maleimide-PEG20K-maleimide-S-GLP-1 receptor binding polypeptide) PGWFLCTAMEADQPVSLT NMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:60), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO: 185 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a terminal cysteine residue with the maleimide;

G-61:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC(S-maleimide-PEG20K-maleimide-S-G LP-1 receptor binding polypeptide)ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTA MEADQPVSLTNMPD EGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO:61), wherein the GLP-1 receptor binding polypeptide has the following sequence: HAibEGTFTSDVSSYLEGQAAKEFIAWLVKGRC(SEQ ID NO:189) and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a terminal cysteine residue with the maleimide;

G-62:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTT SFESAAC (S-maleimide-PEG20K-maleimide-S-GLP-1 receptor binding polypeptide) PGWFLCTAMEADQPVSLT NMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:62), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO: 186 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a terminal cysteine residue with the maleimide;

G-63:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC (S-maleimide-PEG20K-maleimide-S-GLP-1 receptor binding polypeptide) ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPD EGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:63), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO: 187 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a cysteine residue with the maleimide;

G-64:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTT SFESAAC (S-maleimide-PEG20K-maleimide-S-GLP-1 receptor binding polypeptide) PGWFLCTA MEADQPVSLTNMPD EGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:64), wherein the GLP-1 receptor binding polypeptide has the following sequence: HAibEGTFTSDVSSYLEGQAAKEFIAWLVCGRG (SEQ ID NO:190) and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a cysteine residue with the maleimide;

G-65:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC (S-maleimide-PEG20K-maleimide-S-GLP-1 receptor binding polypeptide) ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTA MEADQPVSLTNMPD EGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:65), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO: 188 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a cysteine residue with the maleimide;

G-66:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTT SFESAAC (S-maleimide-PEG20K-maleimide-S-GLP-1 receptor binding polypeptide) PGWFLCTAMEADQPVSLTN-MPDEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:66), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO: 188 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a cysteine residue with the maleimide;

G-67:

(GLP-1 receptor binding polypeptide-S-maleimide-PEG20K-maleimide-S)CRPSGRKSSKMQAFRIWDVNQKTFYL RNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEA VNITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDE GVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO:67), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO: 185 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a terminal cysteine residue with the maleimide;

G-68:

(PEG20 K)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPT TSFE SAAC (S-maleimide-PEG11-maleimide-S-GLP-1 receptor binding polypeptide) PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO: 68), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO: 185 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a terminal cysteine residue with the maleimide;

G-69:

(PEG20 K)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKS GDETRLQLEAVC (S-maleimide-PEG11-maleimide-S-GLP-1 receptor binding polypeptide) ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTA MEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO: 69), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO: 186 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a terminal cysteine residue with the maleimide;

G-70:

(GLP-1 receptor binding polypeptide-S-maleimide-PEG20K-maleimide-S)CRPSGRKSSKMQAFRIWDVNQKTFYL RNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEA VNITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDE GVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:70), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO: 189 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a terminal cysteine residue with the maleimide;

G-71:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC (S-maleimide-PEG11-maleimide-S-GLP-1 receptor binding polypeptide) ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDE GVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:71), wherein the GLP-1 receptor binding polypeptide has the following sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGRC(SEQ ID NO:191) and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a terminal cysteine residue with the maleimide;

G-72:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTT SFESAAC (S-maleimide-PEG11-maleimide-S-GLP-1 receptor binding polypeptide) PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:72), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO: 191 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a terminal cysteine residue with the maleimide;

G-73:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC(S-maleimide-PEG11-maleimide-S-GL P-1 receptor binding polypeptide)ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLT NMPDE GVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO:73), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO:189 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a terminal cysteine residue with the maleimide;

G-74:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTT SFESAAC (S-maleimide-PEG11-maleimide-S-GLP-1 receptor binding polypeptide) PGWFLCTAMEADQPVSLTNM PDEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:74), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO:189 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a terminal cysteine residue with the maleimide;

G-75:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC (S-maleimide-PEG11-maleimide-S-GLP-1 receptor binding polypeptide) ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDE GVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:75), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO: 190 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a cysteine residue with the maleimide;

G-76:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTT SFESAAC (S-maleimide-PEG11-maleimide-S-GLP-1 receptor binding polypeptide) PGWFLCTAMEADQPVSLTNMP DEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:76), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO: 190 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a cysteine residue with the maleimide;

G-77:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC (S-maleimide-PEG11-maleimide-S-GLP-1 receptor binding polypeptide) ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDE GVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:77), wherein the GLP-1 receptor binding polypeptide has the following sequence: HAibEGTFTSDVSSYLEGQAACEFIAWLVKGRG (SEQ ID NO:192) and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a cysteine residue with the maleimide;

G-78:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTT SFESAAC (S-maleimide-PEG11-maleimide-S-GLP-1 receptor binding polypeptide) PGWFLCTAMEADQPVSLTN-MPDEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO: 78), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO: 192 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a cysteine residue with the maleimide;

G-79:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC (S-maleimide-PEG11-maleimide-S-GLP-1 receptor binding polypeptide) ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDE GVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:79), wherein the GLP-1 receptor binding polypeptide has the following sequence: HAEGTFTSDVSSYLEGQAAK[N^(ε)—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-L-Glu)]EFIAWLVRGRC (SEQ ID NO:193) and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a terminal cysteine residue with the maleimide;

G-80:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTT SFESAAC (S-maleimide-PEG11-maleimide-S-GLP-1 receptor binding polypeptide) PGWFLCTAMEADQPVSLTN-MPDEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:80), wherein the GLP-1 receptor binding polypeptide has the sequence of SEQ ID NO: 193 and is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a terminal cysteine residue with the maleimide;

G-81: (SEQ ID NO: 81) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSD AHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK TCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNEC FLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYA PELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCA SLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLL ECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPAD LPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAK TYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEY KFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAED YLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDF AAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSRPSGRK SSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHAL FLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSG PTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE; G-82: (SEQ ID NO: 82) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRPSSGAPPPSGGGGSGGGGSD AHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK TCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNEC FLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYA PELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCA SLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLL ECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPAD LPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAK TYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEY KFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAED YLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDF AAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSRPSGRK SSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHAL FLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSG PTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE; G-83: (SEQ ID NO: 83) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSR PSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFI RSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQED EGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDH VKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMAD CCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYL YEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEG KASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLT KVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHC IAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHP DYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIK QNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCK HPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFS ALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKA TKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL; G-84: (SEQ ID NO: 84) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRPSSGAPPPSGGGGSGGGGSR PSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFI RSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQED EGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDH VKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMAD CCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYL YEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEG KASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLT KVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHC IAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHP DYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIK QNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCK HPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFS ALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKA TKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL;

In the sequences in the section, the structure of C(S-maleimide-(CH₂)—COOH) is shown as follows:

wherein n is an integer from 1 to 25.

In the sequences in the section, the structure of C[S—CH₂—CONH—(CH₂CH₂O)₂—(CH₂)₂—NH—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-Glu)] is shown as follows:

In the sequences in the section, the structure of C[S—CH₂—CONH—(CH₂CH₂O)₂—(CH₂)₂—NH—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-Glu-N-γ-Glu)] is shown as follows:

In the sequences in the section, the structure of C[S-maleimide-(CH₂)₄—NH—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-Glu)] is shown as follows:

In the sequences in the section, the structure of maleimide-PEG11-maleimide is shown as follows:

In the sequences in the section, the structure of maleimide-PEG20K-maleimide is shown as follows:

wherein n is an integer from 0 to 2000.

2. The fusion protein consisting of the insulin receptor binding polypeptide and the interleukin-1 receptor antagonistic protein

The insulin receptor binding polypeptide comprises an A chain and a B chain, in which the amino acid sequence of the A chain is GIVEQC_([3])C_([4])X_(IN8)SIC_([5])SLYQLENYC_([6])X_(IN21)X_(IN22) (SEQ ID NO:380) or GIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N (SEQ ID NO:381);

The amino acid sequence of the B chain is:

X_(IN23-26)HLC_([1])GSHLVEALYLVC_([2])GERGFX_(IN47)X_(IN48)X_(IN49)X_(IN50)X_(IN51)X_(IN52) (SEQ ID NO:3 82), wherein

X_(IN8) is a threonine, histidine, or arginine; X_(IN21) is an alanine, glycine, or asparagine; X_(IN22) is a lysine, a dipeptide of arginine-lysine, or absent; X_(IN23-26) is a tetrapeptide of phenylalanine-valine-asparagine-glutamine, a tripeptide of valine-asparagine-glutamine, a dipeptide of asparagine-glutamine, glutamine, or absent; X_(IN47) is a tyrosine, or phenylalanine; X_(IN48) is a —NH₂, dA-NH₂, tyrosine, or phenylalanine; X_(IN49) is a threonine, asparagine, or absent; X_(IN50) is a lysine, proline, glutamic acid, aspartic acid, or absent; X_(IN51) is a proline, arginine, lysine, glutamic acid, aspartic acid, or absent; X_(IN52) is a threonine, threonine-arginine-arginine, or absent;

[1] to [6] in the compound indicate the Nos. of the cysteines; 3 pairs of disulfide bonds are formed with six cysteines in the compound, in which the A chain and the B chain are connected by two pairs of interchain disulfide bonds, a pair of intrachain disulfide bond is present in the A chain, and the specific positions for the three pairs of the disulfide bonds are: C_([1]) and C_([4]) forming a disulfide bond, C_([2]) and C_([6]) forming a disulfide bond, and C_([3]) and C_([5)] forming a disulfide bond.

In another embodiment, the structure of the amino acid sequence of the single chaininsulin receptor binding polypeptide is:

X_(IN107)HLC_([1])GSX_(IN108)LVEALYLVC_([2])GEX_(IN110)GFX_(IN111)X_(IN112)X_(IN113)X_(IN114)X_(IN115)-C_(L)-GIVEQC_([3])C_([4])X_(IN127)SIC_([5])SLYQLENYC_([6])X_(IN128)X_(IN129) (SEQ ID NO:383), wherein

X_(IN107) is a tetrapeptide of phenylalanine-valine-asparagine-glutamine, a tripeptide of valine-asparagine-glutamine, a dipeptide of asparagine-glutamine, glutamine, a sequence wherein any one of the amino acid residues in the sequence of the tetrapeptide, tripeptide, and dipeptide is substituted with a lysine or arginine, or absent; X_(IN108) is a histidine, phenylalanine, arginine, or glutamine; X_(IN109) is an arginine, alanine, glutamic acid, or aspartic acid; X_(IN110) is a phenylalanine, tyrosine, or histidine; X_(IN111) is a tyrosine, phenylalanine, or absent; X_(IN112) is a threonine, asparagine, or absent; X_(IN113) is a proline, lysine, glutamic acid, aspartic acid, or absent; X_(IN114) is a lysine, proline, arginine, glutamic acid, aspartic acid or absent; X_(IN115) is a threonine, or absent; X_(IN127) is a threonine, histidine, or arginine; X_(IN128) is an alanine, glycine, or asparagine; X_(IN129) is a lysine, a dipeptide of arginine-lysine, or absent; C_(L) is a peptide sequence of 6-60 amino acids, wherein the amino acids are primarily selected from the group consisting of glycine, alanine, serine, threonine and proline.

A suitable linking fragment C_(L) has three characteristics. The first characteristic is that linking fragment is required to have an appropriate length. When the B chain has the full length of thirty amino acids, the linking fragment is better to have no less than six amino acids in length. When the B chain has twenty-five amino acids, the linking fragment is better to have no less than ten amino acids in length. If the length of the linking fragment is too long or too short (shorter than above-mentioned number of the amino acids, or longer than sixty amino acids), the binding capability of the single chain analogue of the insulin receptor tends to decrease. The second is that the linking fragment is preferred to have no secondary structure, so that its spatial conformation can be changed flexibly. The third is that the linking fragment itself has no biological activity and can provide the sites for polypeptide modification, such as, sites for acylation, glycosylation, etc.

The linking fragment C_(L) may include one or more of aspartic acid, glutamic acid, arginine, lysine, cysteine or asparagine residues.C_(L) may comprise 1, 2, 3, 4 of aspartic acid, glutamic acid, arginine, or lysine residues to regulate the charge equilibrium in the sequence of the polypeptide and improve solubility thereof. The sequence can comprise 1, 2, 3, 4, 5 of asparagine and the same amount of serine or threonine, thus constituting a consensus sequence N—X—S/T required for the N-glycosylation (X is an encodable natural amino acid). Furthermore, the peptide can further comprise 1, 2, 3 or 4 of lysine or cysteine residues, of which the amino or mercapto group on the side chain can be linked to a natural or synthetic modifying group such as a fatty acid, polyethylene glycol, an albumin, etc. through a hydrolysable or unhydrolysable bond, thus providing the modified insulin analogue with different physical, chemical, and biological properties.

In accordance with one embodiment, the C-terminal amino acid of the C_(L) may be selected from the group consisting of glycine-lysine, glycine-arginine, arginine-arginine, lysine-lysine, arginine-lysine, lysine-arginine, proline-glutamine-threonine, proline-glutamine-lysine, or proline-glutamine-arginine. In accordance with one embodiment, the C-terminal amino acid of the C_(L) is selected from a lysine or arginine.

In an embodiment, C_(L)is

GX_(IN116)X_(IN117)X_(IN118)X_(IN119)X_(IN120)X_(IN121)X_(IN122)X_(IN123)X_(IN124)X_(IN125)X_(IN126) (SEQ ID NO:384), wherein

X_(IN116) is a lysine, cysteine, serine, or alanine; X_(IN117) is a glycine, lysine, or serine; X_(IN118) is a lysine or serine; X_(IN119) is a lysine or serine; X_(IN120) is a lysine, serine, or alanine; X_(IN121) is a glycine, lysine, arginine, alanine, proline, or absent; X_(IN122) is a glycine, alanine, arginine, lysine, glutamine, proline, or absent; X_(IN123) is an arginine, lysine, glycine, alanine, proline, threonine, glutamine, or absent; X_(IN124) is a proline, glutamine, lysine, glycine, arginine, or absent; X_(IN125) is a glutamine, threonine, lysine, glycine, arginine, or absent; X_(IN126) is a threonine, arginine, lysine, or absent;

In a particular embodiment, C_(L) can be GAGSSSAAAPQT (SEQ ID NO:385), GSGSSSAAAPQT (SEQ ID NO:386), GSGSSSAAPQT (SEQ ID NO:387), GSGSSSAPQT (SEQ ID NO:388), or GSGSSAPQT (SEQ ID NO:389).

In another embodiment, the structure of the amino acid sequence of the single chain insulin receptor binding polypeptide is:

FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFX_(IN111)X_(IN112)X_(IN113)X_(IN114)X_(IN115)-C_(L)-GIV EQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N (SEQ ID NO:390), wherein each of the variables is defined as the above.

The present invention further provides a compound modified on the basis of the insulin receptor binding polypeptide for further increasing the in vivo circulation time for exerting the function of the compound. Said modification is one in which the modified side chain is linked to the α-amino group of the amino acid residue at the N-terminus of the B chain of the double-chain compound according to the present invention or at the N-terminus of the single chain compound according to the present invention, or to the ε-amino group of the lysine present in the double-or single chain compound of the present invention.

In one embodiment, the compound comprises an A chain and a B chain, wherein the amino acid sequence of the A chain is:

(SEQ ID NO: 391) GIVEQC_([3])C_([4])TSIC_([5]) X_(IN412)LX_(IN414)X_(IN415) LX_(IN417)X_(IN418)YC_([6]) X_(IN421)X_(IN422),

the amino acid sequence of the B chain is:

(SEQ ID NO: 392) X_(IN423-426)HLC_([1])GSHLVEALYLVC_([2])GERGFX_(IN447)X_(IN448) X_(IN449)X_(IN450)X_(IN451)X_(IN452)X_(IN453),

[1] to [6] in the compound indicate the Nos of the cysteines; 3 pairs of disulfide bonds are formed with six cysteines in the compound, in which the A chain and the B chain are connected by two pairs of interchain disulfide bonds, a pair of intrachain disulfide bond is present in the A chain, and the specific positions for the three pairs of the disulfide bonds are: C_([1]) and C_([4]) forming a disulfide bond, C_([2]) and C_([6]) forming a disulfide bond, and C_([3]) and C_([5)] forming a disulfide bond;

in which X_(IN412) is a serine or a structure of general formula 1; X_(IN414) is a tyrosine or a structure of general formula 1; X_(IN415) is a glutamine or a structure of general formula 1; X_(IN417) is a glutamic acid or a structure of general formula 1; X_(IN418) is an asparagine or a structure of general formula 1; X_(IN421) is an asparagine, alanine, or glycine; X_(IN422) is a lysine, general formula 3, arginine-general formula 3 or absent; X_(IN423-426) is a glycine-proline-glutamic acid tripeptide, U_(L)-glycine-proline-glutamic acid, a tetrapeptide of phenylalanine-valine-asparagine-glutamine or U_(L)-phenylalanine-valine-asparagine-glutamine; X_(IN447) is a tyrosine or phenylalanine; X_(INT448) is a -NH₂, phenylalanine, tyrosine, or absent; X_(IN449) is a threonine, asparagine, or absent; X_(IN450) is a lysine, arginine, glutamic acid, aspartic acid, proline, or absent; X_(IN451) is a proline, lysine, arginine, glutamic acid, aspartic acid, or absent, or is a structure of general formula 1 or 3; X_(IN452) is a threonine, lysine, or absent, or is a structure of general formula 1 or 3; X_(IN453) is a lysine or absent, or is a structure of general formula 3; U_(L) is defined as herein.

In another embodiment, the structure of the amino acid sequence of the single-stranded compound is:

U_(L)-X_(IN300)HLC_([1])GSHLVEMALVC_([2])GERGFX_(IN301)X_(IN302)X_(IN303)X_(IN304)X_(IN305)X_(IN306)G X_(IN307)X_(IN308)X_(IN309)X_(IN310)X_(IN311)X_(IN312)X_(IN313)X_(IN314)X_(IN315)X_(IN316)X_(IN317)GIVEQC_([3])C_([4])X_(IN318)SIC_([5])X_(IN319)LX_(IN320)X_(IN321)LX_(IN322)X_(IN323)YC_([6])X_(IN324)X_(IN325) (SEQ ID NO:393), in which

X_(IN300) is a tetrapeptide of phenylalanine-valine-asparagine-glutamine, a tripeptide of valine-asparagine-glutamine, a dipeptide of asparagine-glutamine, glutamine, a sequence wherein any one of the amino acid residues in the sequence of the tetrapeptide, tripeptide, and dipeptide is substituted with a lysine or arginine, or absent; X_(IN301) is a phenylalanine, histidine or tyrosine; X_(IN302) is a —NH₂, tyrosine, phenylalanine, or absent; X_(IN303) is a threonine, asparagine, or absent; X_(IN304) is a proline, lysine, glutamic acid, aspartic acid, or absent; X_(IN305) is an aspartic acid, glutamic acid, proline, arginine, lysine, absent or a structure of general formula 1; X_(IN306) is a threonine, a structure of general formula 1, or absent; X_(IN307) is a serine, alanine, glycine, lysine, a structure of general formula 1, or absent; X_(IN308) is a glycine, a structure of general formula 1, or absent; X_(IN309) is a lysine, glycine, serine, a structure of general formula 1, or absent; X_(IN310) is a lysine, glycine, serine, a structure of general formula 1, or absent; X_(IN311) is a lysine, glycine, serine, alanine, a structure of general formula 1, or absent; X_(IN312) is a lysine, arginine, alanine, proline, glycine, a structure of general formula 1, or absent; X_(IN313) is a glycine, alanine, arginine, lysine, glutamine, proline, a structure of general formula 1, or absent; X_(IN314) is an arginine, alanine, proline, threonine, glutamine, glycine, a structure of general formula 1, or absent; X_(IN315) is a proline, glutamine, arginine, glycine, absent, or a structure of general formula 1; X_(IN316) is a glutamine, threonine, arginine, glycine, absent, or a structure of general formula 1; X_(IN317) is a threonine, arginine, lysine, or absent; X_(IN318) is a threonine, histidine, arginine or a structure of general formula 1; X_(IN319) is a serine or a structure of general formula 1; X_(IN320) is a tyrosine or a structure of general formula 1; X_(IN321) is a glutamine or a structure of general formula 1; X_(IN322) is a glutamic acid or a structure of general formula 1; X_(IN323) is an asparagine or a structure of general formula 1; X_(IN324)is an aspartic acid, glycine, alanine, or a structure of general formula 1; X_(IN325) is a lysine, general formula 3, arginine-general formula 3, or absent; U_(L) and the structures of general formulas 1 and 3 are defined as herein.

In another embodiment, the compound has another single chain structure, the amino acid sequence of which is:

U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFX_(IN302)X_(IN303)X_(IN304)X_(IN305)X_(IN306)GX_(IN307)X_(IN308)X_(IN309)X_(IN310)X_(IN311)X_(IN312)X_(IN313)X_(IN314)X_(IN315)X_(IN316)X_(IN317)GIVEQC_([3])C_([4])TSIC_([5])SLYQL ENYC_([6])NX_(IN325) (SEQ ID NO:394), wherein each of the variables is defined as herein.

The insulin receptor binding polypeptide and the interleukin-1 receptor antagonistic protein are able to protect pancreas islet β-cells by similar or distinct mechanisms, thereby treating diabetes mellitus. Accordingly, the fusion protein formed by joining the two polypeptides may attain a therapeutic effect more excellent than that with either of the polypeptides alone, due to the synergistic effect by the two polypeptides. Furthermore, one of the causes for the onset of diabetes is often the inflammation in and around the tissues where the insulin receptor is primarily distributed. The insulin receptor binding polypeptide can serve as a target for the fusion protein and enrich the interleukin-1 receptor antagonistic protein in the tissues, more effectively exerting actions such as anti-inflammation, etc.

The insulin receptor binding polypeptide is linked to the interleukin-1 receptor antagonistic protein in various linkages through an alternative linking group (or spacer group).

(1) The insulin receptor binding polypeptide is lined to the interleukin-1 receptor antagonistic protein in the following form (from left to right corresponding to the amino acid sequence from N-terminus to C-terminus):

the insulin receptor binding polypeptide—linking group (or spacer group)-interleukin-1 receptor antagonistic protein; or

the interleukin-1 receptor antagonistic protein-linking group (or spacer group)-the insulin receptor binding polypeptide;

In one embodiment, the sequence of the fusion protein is

(SEQ ID NO: 395) U_(L)-X_(IN107)HLC_([1])GSX_(IN108)LVEALYLVC_([2])GEX_(IN109) GFX_(IN110)X_(IN111)X_(IN112)X_(IN113)X_(IN114)X_(IN115)-C_(L)- GIVEQC_([3])C_([4])X_(IN127)SIC_([5])SLYQLENYC_([6])X_(IN128) X_(IN129)-L_(j)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQ GPNVNLEEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETR LQLEAVX_(IL84)ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)P GWFLX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE, or (SEQ ID NO: 396) U_(L)-X_(IN107)HLC_([1])GSHLVEALYLVC_([2])GERGFX_(IN110) X_(IN111)X_(IN112)X_(IN113)X_(IN114)X_(IN115)-C_(L)-GIVEQC_([3])C_([4]) TSIC_([5])SLYQLENYC_([6])N-(GGGGS)_(m)-X_(L)-(GGGGS)_(n)-X_(IL0)RP SGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDV VPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVX_(IL84) ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116PGWFLX) _(IL122)T AMEADQPVSLTNMPDEGVMVTKFYFQEDE, or (SEQ ID NO: 397) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKT-C_(L)-GI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N-(GGGGS)_(m)- X_(L)-(GGGGS)_(n)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVA GYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGD ETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PG WFLX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE, or (SEQ ID NO: 398) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKT-C_(L)- GIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N- (GGGGS)_(n)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYL QGPNVNLEEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDET RLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFL X_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE; or (SEQ ID NO: 399) U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNL EEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAV X_(IL84)ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFL X_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE-L_(j)-X_(IN107) HLC_([1])GSX_(IN108)LVEALYLVC_([2])GEX_(IN109)GFX_(IN110) X_(IN111)X_(IN112)X_(IN113)X_(IN114)X_(IN115)-C_(L)-GIVEQC_([3])C_([4]) X_(IN127)SIC_([5])SLYQLENYC_([6])X_(IN128)X_(IN129); or (SEQ ID NO: 400) U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNL EEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAV X_(IL84)ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWF LX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE-(GGGGS)_(m)- X_(L)-(GGGGS)_(n)-X_(IN107)HLC_([1])GSHLVEALYLVC_([2])GER GFX_(IN110)X_(IN111)X_(IN112)X_(IN113)X_(IN114)X_(IN115)- C_(L)-GIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])X_(IN128) X_(IN129), or (SEQ ID NO: 401) U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNL EEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEA VNITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFLX_(IL122) TAMEADQPVSLTNMPDEGVMVTKFYFQEDE-(GGGGS)_(m)-X_(L)- (GGGGS)_(n)-FVNQHLC_([1])GSHLVEALYLVC_([2])GE RGFFYTPKT-C_(L)-GIVEQC_([3])C_([4])TSIC_([5]) SLYQLENYC_([6])N; or (SEQ ID NO: 402) U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNL EEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAV NITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFLX_(IL122) TAMEADQPVSLTNMPDEGVMVTKFYFQEDE-(GGGGS)_(n)-FVNQHL C_([1]GSHLVEALYLVC) _([2])GERGFFYTPKT-C_(L)-GIVEQC_([3]) C_([4])TSIC_([5]SLYQLENYC) _([6])N; or (SEQ ID NO: 403) U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNL EEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEA VX_(IL84)ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGW FLX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE-(GGGGS)_(m)- X_(L)-(GGGGS)_(n)-FVNQHLCGSHLVEALYLVCGERGFFYTPKTG SGSSSAAAPQTGIVEQCCTSICSLYQLENYCN;

in the above-mentioned sequences, m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as herein;

[1] to [6] indicate the Nos. of the cysteines; 3 pairs of disulfide bonds are formed by six cysteines in the compound, in which the A chain and the B chain are connected by two pairs of interchain disulfide bonds, a pair of intrachain disulfide bond is present in the A chain, and the specific positions for the three pairs of the disulfide bonds are: C_([1]) and C_([4]) forming a disulfide bond, C_([2]) and C_([6]) forming a disulfide bond, and C_([3]) and C_([5]) forming a disulfide bond.

The fusion protein consisting of the insulin receptor binding polypeptide and the interleukin-1 receptor antagonistic protein is selected from:

IN-1:

(SEQ ID NO: 85) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPK TGSGSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6]) NRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKID VVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDK RFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTK FYFQEDE; IN-2: (SEQ ID NO: 86) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKTG SGSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6]) NRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVV PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFA FIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYF QEDE; IN-3: (SEQ ID NO: 87) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKTG SGSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6]) NRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDV VPIEPHALFLGIHGGKMSLSSVKSGDETRLQLEAVNITDLSENRKQDKR FAFIRSDSGPTTSFESAASPGWFLSTAMEADQPVSLTNMPDEGVMVTK FYFQEDE; IN-4: (SEQ ID NO: 88) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKTGSGSSSAAAPQT GIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N GGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEE KIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRK QDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVM VTKFYFQEDE; IN-5: (SEQ ID NO: 89) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKTGS GSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6]) NGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEE KIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQ DKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMV TKFYFQEDE; IN-6: (SEQ ID NO: 90) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKTGSGSSS AAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NG GGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKI DVVPIEPHALFLGIHGGKMSLSSVKSGDETRLQLEAVNITDLSENRKQDK RFAFIRSDSGPTTSFESAASPGWFLSTAMEADQPVSLTNMPDEGVMVTK FYFQEDE; IN-7: (SEQ ID NO: 91) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKTGSGSS SAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NG GGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVN LEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSEN RKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGV MVTKFYFQEDE; IN-8: (SEQ ID NO: 92) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKTGSGSSS AAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N GGGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPN VNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDL SENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMP DEGVMVTKFYFQEDE; IN-9: (SEQ ID NO: 93) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKTGSGSSSAAA PQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NG GGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVN LEEKIDVVPIEPHALFLGIHGGKMSLSSVKSGDETRLQLEAVNITDLSEN RKQDKRFAFIRSDSGPTTSFESAASPGWFLSTAMEADQPVSLTNMPDEGV MVTKFYFQEDE; IN-10: (SEQ ID NO: 94) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKTGSGSSSAA APQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NG GGGSGGGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQ GPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNIT DLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNM PDEGVMVTKFYFQEDE; IN-11: (SEQ ID NO: 95) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKTGSGSSSA AAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NG GGGSGGGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQ GPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNIT DLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNM PDEGVMVTKFYFQEDE; IN-12: (SEQ ID NO: 96) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKTGSGSSSAA APQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NGG GGSGGGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQG PNVNLEEKIDVVPIEPHALFLGIHGGKMSLSSVKSGDETRLQLEAVNITD LSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLSTAMEADQPVSLTNMP DEGVMVTKFYFQEDE; IN-13: (SEQ ID NO: 97) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAAAPQTG IVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYF QEDE; IN-14: (SEQ ID NO: 98) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAAAPQT GIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NR PSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFA FIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFY FQEDE; IN-15: (SEQ ID NO: 99) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAAAPQT GIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NR PSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMSLSSVKSGDETRLQLEAVNITDLSENRKQDKRFAFI RSDSGPTTSFESAASPGWFLSTAMEADQPVSLTNMPDEGVMVTKFYFQ EDE; IN-16: (SEQ ID NO: 100) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAAAPQTG IVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N GGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEK IDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQD KRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTK FYFQEDE; IN-17: (SEQ ID NO: 101) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAAAPQTGI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NG GGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEK IDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRK QDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGV MVTKFYFQEDE; IN-18: (SEQ ID NO: 102) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAAAPQTGI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NG GGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEK IDVVPIEPHALFLGIHGGKMSLSSVKSGDETRLQLEAVNITDLSENRKQ DKRFAFIRSDSGPTTSFESAASPGWFLSTAMEADQPVSLTNMPDEGVMV TKFYFQEDE; IN-19: (SEQ ID NO: 103) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAAAPQTGI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NGG GGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNL EEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENR KQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVM VTKFYFQEDE; IN-20: (SEQ ID NO: 104) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAAAPQTGI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NG GGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNV NLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLS ENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPD EGVMVTKFYFQEDE; IN-21: (SEQ ID NO: 105) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAAAPQTGI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NG GGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNV NLEEKIDVVPIEPHALFLGIHGGKMSLSSVKSGDETRLQLEAVNITDLS ENRKQDKRFAFIRSDSGPTTSFESAASPGWFLSTAMEADQPVSLTNMPD EGVMVTKFYFQEDE; IN-22: (SEQ ID NO: 106) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAAAPQTGI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NG GGGSGGGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQ GPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNIT DLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNM PDEGVMVTKFYFQEDE; IN-23: (SEQ ID NO: 107) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAAAPQTGI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NG GGGSGGGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQ GPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNIT DLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNM PDEGVMVTKFYFQEDE; IN-24: (SEQ ID NO: 108) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAAAPQT GIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NG GGGSGGGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYL QGPNVNLEEKIDVVPIEPHALFLGIHGGKMSLSSVKSGDETRLQLEAVN ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLSTAMEADQPVSL TNMPDEGVMVTKFYFQEDE; IN-25: (SEQ ID NO: 109) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKTGSGSSSAAA PQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NG GGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNV NLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLS ENRKQDKRFAFIRSDSGPTTSFESAAC[S-maleimide- (CH₂)₄—NH—(N^(α)—(HOOC(CH₂)₁₄CO)- γ-Glu)]PGWF LCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); IN-26: (SEQ ID NO: 110) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKTGSGSSSAAA PQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NG GGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVN LEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAV C[S—CH₂—CONH—(CH₂CH₂O)₂—(CH₂)₂—NH—(N^(α)—(HOOC(CH₂)₁₄ CO)-γ-Glu)]ITDLSENRKQDKRFAFIRSDSGPTTSFES AASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); IN-27: (SEQ ID NO: 111) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFEGSGSSSAAAPQTGI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NG GGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVN LEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSEN RKQDKRFAFIRSDSGPTTSFESAAC(S—CH₂—CONH-PEG20K)PGWFLC TAMEADQPVSLTNMPDEGVMVTKF YFQEDE (S is the sulphur atom on the side chain of a cysteine residue); IN-28: (SEQ ID NO: 112) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFEGSGSSSAAAPQTG IVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NG GGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVN LEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC (S- maleimide-(CH₂)₁₅—COOH)ITDLSENRKQDKRFAFIRS DSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDE GVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue); IN-62 (SEQ ID NO: 146) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVV PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKR FAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTK FYFQEDEGGGGSGGGGSFVNQHLC_([1])GSHLVEALYLVC_([2]) GERGFFYTPKTGSGSSSAAAPQTGIVEQC_([3])C_([4])TS IC_([5])SLYQLENYC_([6])N; IN-63 (SEQ ID NO: 147) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DEGGGGSGGGGSFVNQHLC[1]GSHLVEALYLVC_([2])GERGFFYTPKTG SGSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])S LYQLENYC_([6])N; IN-64: (SEQ ID NO: 148) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMSLSSVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAASPGWFLSTAMEADQPVSLTNMPDEGVMVTKFYFQE DEGGGGSGGGGSFVNQHLC_([1])GSHLVEALYLVC_([2])GERGF FYTPKTGSGSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SL YQLENYC_([6])N; IN-65 (SEQ ID NO: 149) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVCITDLSENRKQDKRFAF IRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DEGGGGSGGGGSFVNQHLC[1]GSHLVEALYLVC_([2])GERGFFYTPKTG SGSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])S LYQLENYC_([6])N; IN-66 (SEQ ID NO: 150) MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVV PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFA FIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQ EDEGGGGSCGGGGSGGGGSFVNQHLC_([1])GSHLVEALYLVC_([2])G ERGFFYTPKTGSGSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLY QLENYC_([6])N; IN-67 (SEQ ID NO: 151) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKTGSGSSS AAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENY C_([6])NGGGGSGGGGSCGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRN NQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETR LQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEA DQPVSLTNMPDEGVMVTKFYFQEDE;

In the sequences of this section, [1] to [6] indicate the Nos. of the cysteines; 3 pairs of disulfide bonds are formed by six cysteines in the compound, in which the A chain and the B chain are connected by two pairs of interchain disulfide bonds, a pair of intrachain disulfide bond is present in the A chain, and the specific positions for the three pairs of the disulfide bonds are: C_([1]) and C_([4]) forming a disulfide bond, C_([2]) and C_([6]) forming a disulfide bond, and C_([3]) and C_([5]) forming a disulfide bond.

In one embodiment, the insulin receptor binding polypeptide, the interleukin-1 receptor antagonistic protein, and a biomacromolecule form a single chain compound, for which the structure is (from left to right corresponding to the amino acid sequence from N-terminus to C-terminus):

the insulin receptor binding polypeptide—linking group (or spacer group)-biomacromolecule-linking group (or spacer group)-interleukin-1 receptor antagonistic protein;

the insulin receptor binding polypeptide—linking group (or spacer group)-interleukin-1 receptor antagonistic protein-linking group (or spacer group)-the biomacromolecule;

the biomacromolecule-linking group (or spacer group)-the insulin receptor binding polypeptide—linking group (or spacer group)-interleukin-1 receptor antagonistic protein;

the biomacromolecule-linking group (or spacer group)-the interleukin-1 receptor antagonistic protein-linking group (or spacer group)-the insulin receptor binding polypeptide;

the interleukin-1 receptor antagonistic protein-linking group (or spacer group)-biomacromolecule-linking group (or spacer group)-the insulin receptor binding polypeptide; or

the interleukin-1 receptor antagonistic protein-linking group (or spacer group)-the insulin receptor binding polypeptide - linking group (or spacer group)-the biomacromolecule;

in which the biomacromolecule may be albumin, IgG Fc, etc.

In one embodiment, the sequence of the fusion protein comprising the human albumin is

(SEQ ID NO: 404) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFA KTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNE CFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFY APELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKC ASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDL LECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPA DLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLA KTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAE DYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPK EFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDD FAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL-(GGGGS)_(m)-FVNQH LC_([1])GSHLVEALYLVC_([2])GERGFFYTPKT-C_(L)- GIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N- (GGGGS)_(n)-RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNV NLEEKIDVVPIEPHALFLGIHGGKMX_(IL66LSX) _(IL69)VKSGDETRLQL EAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWF LX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE; or (SEQ ID NO: 499) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFA KTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNE CFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFY APELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKC ASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDL LECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPA DLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLA KTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAE DYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPK EFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDD FAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL-(GGGGS)_(m)RP SGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIE PHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVN ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFLX_(IL122)T AMEADQPVSLTNMPDEGVMVTKFYFQEDE-(GGGGS)_(N)-FVNQHL C_([1])GSHLVEALYLVC_([2])GERGFFYTPKT-C_(L)- GIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 405) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKT-C_(L)- GIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N- (GGGGS)_(m)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFE DHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEM ADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKK YLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRD EGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTD LTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKS HCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARR HPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNL IKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKC CKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPC FSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKP KATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL- (GGGGS)_(n)-RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQG PNVNLEEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDET RLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGW FLX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE; or (SEQ ID NO: 406) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKT-C_(L)- GIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N- (GGGGS)_(m)-RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGP NVNLEEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKS GDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)P GWFLX_(IL22)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE-(GGGGS)_(n)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFA KTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNE CFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFY APELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKC ASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDL LECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPA DLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLA KTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAE DYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPK EFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDD FAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL; or (SEQ ID NO: 407) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKTG SGSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLEN YC_([6])NGGGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQ LVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69) VKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESA AX_(IL116PGWFLX) _(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE GGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFE DHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETY GEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHD NEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAAC LLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRF PKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDS ISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKN YAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPH ECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKV PQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLC VLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFT FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFV EKCCKADDKETCFAEEGKKLVAASQAALGL; or (SEQ ID NO: 408) X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNL EEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGD ETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116) PGWFLX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE- (GGGGS)_(m)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCP FEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETY GEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEET FLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEV SKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCE KPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGM FLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFK PLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVS RNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKC CTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKK QTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKK LVAASQAALGL-(GGGGS)_(n)-FVNQHLC_([1])GSHLV EALYLVC_([2])GERGFFYTPKT-C_(L)-GIVEQC_([3])C_([4]) TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 409) X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNL EEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGD ETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116) PGWFLX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE- (GGGGS)_(m)-FVNQHLC_([1])GSHLVEALYLVC_([2]) GERGFFYTPKT-C_(L)-GIVEQC_([3])C_([4])TSIC_([5])SL YQLENYC_([6])N-(GGGGS)_(n)-DAHKSEVAHRFKDLG EENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDK SLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLP RLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYK AAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAF KAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADL AKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADF VESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLE KCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNAL LVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVV LNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAE TFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAF VEKCCKADDKETCFAEEGKKLVAASQAALGL;

in each of the above-mentioned fusion proteins, m and n are each 0, 1, 2, 3, 4, 5, or 6 and each of other variables is defined as herein;

IN-68:

(SEQ ID NO: 152) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFA KTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNE CFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFY APELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKC ASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDL LECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPA DLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLA KTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAE DYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPK EFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDD FAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSFVNQH LC_([1])GSHLVEALYLVC_([2])GERGFFYTPKTGS GSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5]) SLYQLENYC_([6])NGGGGSGGGGSRPSGRKSSKMQAFRIWDVN QKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLS CVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPG WFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE; IN-69: (SEQ ID NO: 153) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFA KTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNE CFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFY APELLI-FAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK CASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGD LLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMP ADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRL AKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLG EYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCA EDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVP KEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMD DFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSRPSG RKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPH ALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSD SGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDEGG GGSGGGGSFVNQHLC_([1])GSHLVEALYLVC_([2])GERGFF YTPKTGSGSSSAAAPQTGIVEQC_([3])C_([4])TS IC_([5])SLYQLENYC_([6])N; IN-70: (SEQ ID NO: 154) MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVV PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFA FIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQ EDEGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFE DHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEM ADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKK YLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRD EGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTD LTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKS HCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARR HPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQN LIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSK CCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRP CFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHK PKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLG GGGSGGGGSFVNQHLC_([1])GSHLVEALYLVC_([2])GE RGFFYTPKTGAGSSSAAAPQTGIVEQC_([3])C_([4])TS IC_([5])SLYQLENYC_([6])N.

(2) A dimeric protein/cross-linking protein of the interleukin-1 receptor antagonistic protein—the linking or spacer group-the insulin receptor binding polypeptide

If the insulin receptor binding polypeptide is linked to the linking group via a chemical reaction and further linked to the interleukin-1 receptor antagonistic protein, there is of regularity somewhat in selecting the reactive functional groups and reactive sites on the insulin receptor binding polypeptide.

The human natural insulin has just one lysine at B29. The single chain insulin receptor binding polypeptide is linked to the linking group through the N-terminal α-amino group, the ε-amino groups on the side chains of the intrachain or terminal lysines or the mercapto groups on the side chains of the intrachain or terminal cysteines. The double-chain insulin receptor binding polypeptide is linked to the linking group through the N-terminal α-amino group of the B chain, the ε-amino group on the side chain of the lysine or the mercapto group on the side chain of the cysteine at the N- or C-terminus of the B chain, or the amino group on the side chain of the lysine or the mercapto group on the side chain of the cysteine at the C-terminus of the A chain.

In one embodiment, the sequence of the fusion protein is

(SEQ ID NO: 410) U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQ LVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMX_(IL66)LS X_(IL69)VKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGP TTSFESAAC(the linking group-the insulin receptor bindingpolypeptide)PGWFLX_(IL122)TAMEADQPVSLTNM PDE GVMVTKFYFQEDE; or (SEQ ID NO: 411) U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQ LVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMX_(IL66)LS X_(IL69)VKSGDETRLQLEAVC(the linking group-the insulin receptor binding polypeptide)ITDLSENRKQDKRF AFIRSDSGPTTSFESAASPGWFLX_(IL122)TAMEADQPVSLTNM PDEGVMVTKFYFQEDE; or (SEQ ID NO: 412) (the insulin receptor binding polypeptide-the linking group)-C_(IL0)-X_(IL0)RPSGRKSSKMQAFRIWD VNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGG KMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVX_(IL84) ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGW FLX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE, or (SEQ ID NO: 413) U_(L)-X_(IL10)RPSGRKSSKMQAFRIWDVNQKTFYLRNN QLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMX_(IL66)L SX_(IL69)VKSGDETRLQLEAVX_(IL84)ITDLSENRKQD KRFAFIRSDSGPTTSFESAAX_(IL116)PGWFLX_(IL122) TAMEADQPVSLTNMPDEGVMVTKFYFQEDEC_(IL153) (the linking group-the insulin receptor binding polypeptide),

In the individual fusion proteins, each of the variables is defined as herein.

In one embodiment, one terminus of the linking group, which is a N-hydroxyl succinimide (NHS ester), is reacted with the amino group on the side chain of a lysine in the insulin receptor binding polypeptide, while the other terminus thereof, which is a modifying group activated by maleimide or iodoethylamide, is reacted with the cysteine at position 0, 6, 8, 9, 84, 116, 141, or 153 in the interleukin-1 receptor antagonistic protein, thereby forming a covalent linkage. In one embodiment, one terminus of the linking group, which is a modifying group activated by maleimide or iodoethylamide, is reacted with the cysteine at position 0, 6, 8, 9, 84, 116, 141, or 153 in the interleukin-1 receptor antagonistic protein to form a covalent linkage, while the other terminus thereof, which is an aldehyde group, is reacted with the amino group at N-terminus of the insulin receptor binding polypeptide, thereby forming a covalent linkage via reductive amination. In one embodiment, one terminus of the linking group, which is a NHS ester, is reacted with the amino group on the side chain of a lysine in the insulin receptor binding polypeptide, while the other terminus thereof, which is an aldehyde group, is reacted with the amino group at N-terminus of theinterleukin-1 receptor binding polypeptide, thereby forming covalent linkages via reductive amination.

The insulin receptor binding polypeptide has the single-or double-chain structure set forth in the invention. In one embodiment, the insulin receptor binding polypeptide is the human natural insulin, the human natural insulin desB30T, lispro insulin, aspart insulin, glulisine insulin, etc. The α-amino group at the N-terminus of the B chain or the ε-amino group on the side chain of an intrachain lysine may be reacted with the NHS ester at one terminus of the linking group. In one embodiment, the insulin receptor binding polypeptide is a modified conjugate, for example, glargine insulin, insulin detemir, etc.

The single chain insulin receptor binding polypeptide may have one of the following sequences:

(SEQ ID NO: 414) U_(L)-X_(IN107)HLC_([1])GSX_(IN108)LVEALYLVC_([2])GEX_(IN109)GFX_(IN110)X_(IN111)X_(IN112)X_(IN113)X_(IN114) X_(IN115)-C_(L)-GIVEQC_([3])C_([4])X_(IN127)SICSLYQLENYC_([6])X_(IN128)X_(IN129); or (SEQ ID NO: 415) U_(L)-X_(IN107)HLC_([1])GSHLVEALYLVC_([2])GERGFX_(IN110)X_(IN111)X_(IN112)X_(IN113)X_(IN114)X_(IN115)- C_(L)-GIVEQC_([3])C_([4])TSICSLYQLENYC_([6])X_(IN128)X₁₂₉; or (SEQ ID NO: 416) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)T-C_(L)-GIVEQC_([3])C_([4])TSI C_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 417) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)TGKGSSSAAAPQTGI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 418) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)TGSGKSSAAAPQTGI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 419) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)TGSGSKSAAAPQTGI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 420) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)TGSGSSKAAAPQTGI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 421) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)TGSGSSSKAAPQTGIV EQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 422) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)TGSGSSSAKAPQTGIV EQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 423) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)TGSGSSSAAKPQTGIV EQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 424) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)TGSGSSSAAAPQTGIV EQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NK; or (SEQ ID NO: 425) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)TGSGSSSAAAPQTGIVEQ C_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 426) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAAKPQTGIVEQC_([3])C_([4])T SIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 427) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAKAPQTGIVEQC_([3])C_([4])T SIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 428) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSKAAPQTGIVEQC_([3])C_([4])T SIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 429) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAAAPQTGIVEQC_([3])C_([4])T SIC_([5])SLYQLENYC_([6])NK; or (SEQ ID NO: 430) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5]) SLYQLENYC_([6])N;

The variables in the individual peptides are defined as herein;

The single chain insulin receptor binding polypeptide is linked to the linking group through the α-amino group at the N-terminus or the ε-amino group on the side chain of a lysine.

The interleukin-1 receptor antagonistic protein for forming a dimeric protein/cross-linking protein may be

(SEQ ID NO: 431) U_(L)-RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKI DVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAV X_(IL84)ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWF LX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE; or (SEQ ID NO: 432) U_(L)-CRPSGRKSSKMQAPRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEE KIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVN ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLX_(IL122)TAMEADQ PVSLTNMPDEGVMVTKFYFQEDE; or (SEQ ID NO: 433) U_(L)-RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEK IDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVNIT DLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLX_(IL122)TAMEADQP VSLTNMPDEGVMVTKFYFQEDEC; each of the variables in the individual proteins is defined as herein.

The insulin receptor binding polypeptide-a linking or spacer group-the interleukin-1 receptor antagonistic protein may be:

(the insulin receptor binding polypeptide -the linking group)RPSGRKSS KMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEP HALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSG PTTSFESAAX_(IL116)PGWFLX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:434); or

(the insulin receptor binding polypeptide -the linking group)CRPSGRKSSKMQAPRIVVDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDS GPTTSFE,SAASPGWFLX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:435); each of the variables in the individual proteins is defined as herein.

The sequences of the dimeric protein/cross-linking protein of the insulin receptor binding polypeptide and the interleukin-1 receptor antagonistic protein are selected from:

IN-29:

FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPRTGK(N^(ε)-PEG12-maleimide-IL-1 ra) GSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQ LENYC_([6])N(SEQ ID NO:113), wherein the sequence of the IL-1ra is:

(SEQ ID NO: 194) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE,

the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with the amino group on the side chain of a lysine in the insulin receptor binding polypeptide, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 116 underlined in the IL-1ra.

IN-30:

FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPRTGK(N^(ε)-PEG12-maleimide-the interleukin-1 receptor antagonistic protein) GSSSAAAPQT GIVEQC_([3])C_([4])TSIC_([5])SLYQ LENYC_([6])N; (SEQ ID NO: 114)

in which the sequence of the interleukin-1 receptor antagonistic protein is as follows:

(SEQ ID NO: 195) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVCITDLSENRKQDKRFAF IRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE,

The N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with the amino group on the side chain of a lysine in the insulin receptor binding polypeptide, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 84 underlined in the interleukin-1 receptor antagonistic protein.

IN-31:

FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPRTGK(N^(ε)-PEG20K-maleimide-IL-1ra) GSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQ LENYC_([6])N(SEQ ID NO:115), in which the sequence of IL-1ra is SEQ ID NO:194 and the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG20K-NHS, is reacted with the amino group on the side chain of a lysine in the insulin receptor binding polypeptide, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 116 underlined in the IL-1ra.

IN-32:

FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPRTGK(N^(ε)-PEG20K-maleimide-the interleukin-1 receptor antagonistic protein) GSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N(SEQ ID NO:116), in which the sequence of the interleukin-1 receptor antagonistic protein is SEQ ID NO:195, and the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG20K-NHS, is reacted with the amino group on the side chain of a lysine in the insulin receptor binding polypeptide, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 84 underlined in the interleukin-1 receptor antagonistic protein.

IN-33:

F[N^(α)—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-L-Glu]VNQHLC_([1])GSHLVEALYLVC_([2])GERGFF YTPRTGK(N^(ε)-PEG12-maleimide-IL-1ra) GSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQ LENYC_([6])N(SEQ ID NO:117); in which the sequence of IL-1ra is SEQ ID NO:194 and the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with the amino group on the side chain of a lysine in the insulin receptor binding polypeptide, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 116 underlined in the IL-1ra.

IN-34:

F[N^(α)—(N^(α)—-(HOOC(CH₂)₁₆CO)-γ-L-Glu)]VNQHLC_([1])GSHLVEALYLVC_([2])GERGFF YTPRTGK(N^(ε)-PEG12-maleimide-the interleukin-1 receptor antagonistic protein) GSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N(SEQ ID NO: 118), in which the sequence of the interleukin-1 receptor antagonistic protein is SEQ ID NO:195, and the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with the amino group on the side chain of a lysine in the insulin receptor binding polypeptide, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 84 underlined in the interleukin-1 receptor antagonistic protein.

IN-35:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTT SFESAAC(S-maleimide-PEG12-human insulin)PGWFLCTAMEAD QPVSLTNMPDEGVMVTKFY FQEDE (S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO:119);

the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with α-amino group at the N-terminus of the B chain of the human insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 116 in the IL-1ra.

IN-36:

(SEQ ID NO: 120) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAAC(S-maleimide-PEG12-the insulin rec eptor bindingpolypeptide)PGWFLCTAMEADQPVSLTNMPDEGV MV TKFYFQEDE (S is the sulphuratom on the side chain of a cysteine residue),

the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with the α-amino group at the N-terminus of the insulin receptor binding polypeptide N^(εB29)—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-Glu)des(B30)-the B chain of the human insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 116 in the IL-1ra.

IN-37:

(SEQ ID NO: 121) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAAC(S-maleimide-PEG20K-human insulin)PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atomon the side chain of a cysteine residue);

the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG20K-NHS, is reacted with α-amino group at the N-terminus of the B chain of the human insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 116 in the IL-1ra.

IN-38:

(SEQ ID NO: 122) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC(S-maleimide-PEG 12-human insulin)ITDLSENRK QDKRFAFIRSDSGPTTSFESAA SPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue);

in which the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with α-amino group at the N-terminus of the B chain of the human insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 84 in the interleukin-1 receptor antagonistic protein.

IN-39:

(SEQ ID NO: 123) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC(S-maleimide-PEG 20K-humaninsulin)ITDLSEN-RKQDKRFAFIRSDSGPTTSFESAA SPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue);

in which the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG20K-NHS, is reacted with α-amino group at the N-terminus of the B chain of the human insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 84 in the interleukin-1 receptor antagonistic protein.

IN-40:

(SEQ ID NO: 124) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC(S-maleimide-PEG 12-the insulinreceptor binding polypeptide) ITDLSE NRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEAD QPVSLTNMPDE GVMV TKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue);

in which the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with the α-amino group at the N-terminus of the insulin receptor binding polypeptide N^(εB29)—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-Glu)des(B30)-the B chain of the human insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 84 in the interleukin-1 receptor antagonistic protein.

IN-41:

(SEQ ID NO: 125) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAAC(S-maleimide-PEG12-human insulin d esB30T)PGWFLCTAMEADQPVSLTNMPDEGV MVTK FYFQEDE (S is the sulphur atom on theside chain of a cysteine residue);

in which the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with the ε-amino group of a lysine in the human insulin desB30T B29, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 116 in the IL-1ra.

IN-42:

(SEQ ID NO: 126) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAAC(S-maleimide-PEG20K-human insulin desB30T)PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on theside chain of a cysteine residue);

in which the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with the ε-amino group of a lysine in the human insulin desB30T B29, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 116 in the IL-1ra.

IN-43:

(SEQ ID NO: 127) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC(S-maleimide-PEG 12-human insulindesB30T) ITDLSENRKQDKRFAFIRSDSGPT TSFESAASPG WFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue),

in which the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with the ε-amino group of a lysine in the human insulin desB30T B29, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 84 in the interleukin-1 receptor antagonistic protein.

IN-44:

(SEQ ID NO: 128) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC(S-maleimide-PEG 20K-human insulindesB30T)ITDLSENRKQDKRFAFIRSDSGPT TSFESAASPG WFLCTAMEADQPVSLTNMPDEGVMV TKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue),

in which the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG20K-NHS, is reacted with the ε-amino group of a lysine in the human insulin desB30T B29, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 84 in the interleukin-1 receptor antagonistic protein.

IN-45:

FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPRTGSGSSSAAAPQTGIVEQC_([3])C₁₄TSIC_([5])SLYQLENYC_([6])NK (N^(ε)-PEG12-maleimide-IL-1ra) (SEQ ID NO: 129), wherein the sequence of the IL-1ra is SEQ ID NO:194;

in which the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with the ε-amino group on the side chain of a lysine at the C-terminus of the single chain insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 116 underlined in the IL-1ra.

IN-46:

FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPRTGSGSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NK (N^(ε)-PEG20K-maleimide-IL-1ra)(SEQ ID NO: 130), wherein the sequence of the IL-1ra is SEQ ID NO:194;

the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG20K-NHS, is reacted with the ε-amino group on the side chain of a lysine at the C-terminus of the single chain insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 116 underlined in the IL-1ra.

IN-47:

FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPRTGSGSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NK (N^(ε)-PEG12-maleimide-the interleukin-1 receptor antagonistic protein) (SEQ ID NO: 131), the sequence of the interleukin-1 receptor antagonistic protein is SEQ ID NO:195;

in which the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with the ε-amino group on the side chain of a lysine at the C-terminus of the single chain insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 84 underlined in the interleukin-1 receptor antagonistic protein.

IN-48:

FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPRTGSGSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NK (N^(ε)-PEG20K-maleimide-the interleukin-1 receptor antagonistic protein)(SEQ ID NO: 132), the sequence of the interleukin-1 receptor antagonistic protein is SEQ ID NO:195, and the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG20K-NHS, is reacted with the ε-amino group on the side chain of a lysine at C-terminus of the single chain insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 84 underlined in the interleukin-1 receptor antagonistic protein.

IN-49:

F(N^(α)-PEG12-maleimide-IL-1ra)VNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPRT GSG SSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N(SEQ ID NO:133), wherein the sequence of the IL-1ra is

(SEQ ID NO: 196) CRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVV PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFA FIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQ EDE;

the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with α-amino group at the N-terminus of the single chain insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at the N-terminus of the interleukin-1 receptor antagonistic protein.

IN-50:

F(N^(α)-PEG20K-maleimide-IL-1ra) VNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPRT GSGSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N-(SEQ ID NO: 134), wherein the sequence of the IL-1ra is SEQ ID NO:196;

the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG20K-NHS, is reacted with the α-amino group at the N-terminus of the single chain insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at the N-terminus of the interleukin-1 receptor antagonistic protein.

IN-51:

FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPRTGK(N^(ε)-PEG12-maleimide-the interleukin-1 receptor antagonistic protein GSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6]) N-(SEQ ID NO: 135), in which the sequence of the interleukin-1 receptor antagonistic protein is SEQ ID NO: 196;

the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with the ε-amino group on the side chain of a lysine in the single chain insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at the N-terminus of the interleukin-1 receptor antagonistic protein.

IN-52:

FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPRTGK(N^(ε)-PEG20K-maleimide-the interleukin-1 receptor antagonistic protein) GSSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLEN-YC_([6]) N(SEQ ID NO: 136);

in which the sequence of the interleukin-1 receptor antagonistic protein is SEQ ID NO: 196;

in which the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG20K-NHS, is reacted with the ε-amino group on the side chain of a lysine in the single chain insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at the N-terminus of the interleukin-1 receptor antagonistic protein.

IN-53:

FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPRTGSGSSSAK(N^(ε)-PEG12-maleimide-the interleukin-1 receptor antagonistic protein)APQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N(SEQ ID NO: 137), in which the sequence of the interleukin-1 receptor antagonistic protein is SEQ ID NO: 196;

in which the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with the ε-amino group on the side chain of a lysine in the single chain insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at the N-terminus of the interleukin-1 receptor antagonistic protein. IN-54:

FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPRTGSGSSSAK(N^(ε)-PEG20K-male imide-the interleukin-1 receptor antagonistic protein)APQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N(SEQ ID NO: 138), in which the sequence of the interleukin-1 receptor antagonistic protein is SEQ ID NO: 196;

in which the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG20K-NHS, is reacted with the ε-amino group on the side chain of a lysine in the single chain insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at the N-terminus of the interleukin-1 receptor antagonistic protein.

IN-55:

FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAK(N^(ε)-PEG12-maleimide-IL-1ra) APQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N(SEQ ID NO: 139), in which the sequence of the IL-1ra is SEQ ID NO:194,

the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with the amino group on the side chain of a lysine in the insulin receptor binding polypeptide, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 116 underlined in the IL-1ra.

IN-56:

FVNQHLC_([1])GSHLVEALYLVC_([2])GERGEFGSGSSSAK (N^(ε)-PEG12-maleimide-the interleukin-1 receptor antagonistic protein) APQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N(SEQ ID NO:140), in which the sequence of the interleukin-1 receptor antagonistic protein is SEQ ID NO:195, and the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with the amino group on the side chain of a lysine in the insulin receptor binding polypeptide, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at position 84 underlined in the interleukin-1 receptor antagonistic protein.

IN-57:

C(S-maleimide-PEG12-human insulin) RPSGRKSSKMQAFRIWD VNQKTFYL RNNQLVA GYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSE NRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKF YFQEDE (S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO:141),

the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with α-amino group at the N-terminus of the B chain of the human insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at the N-terminus of the interleukin-1 receptor antagonistic protein.

IN-58:

C(S-maleimide-PEG12-the insulin receptor binding polypeptide) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALF LGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAA SPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO:142),

the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with the α-amino group at the N-terminus of the insulin receptor binding polypeptide N^(εB29)—(N^(α)—(HOOC(CH₂)₁₄CO) -γ-Glu)des(B30)-the B chain of the human insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at the N-terminus of the interleukin-1 receptor antagonistic polypeptide.

IN-59:

C(S-maleimide-PEG12-human insulin desB30T) RPSGRKSSKMQAFRIWDVNQKTFYL RNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEA VNITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDE GVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO:143),

the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with the ε-amino group on the side chain of a lysine in the human insulin desB30T B29, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at the N-terminus of the interleukin-1 receptor antagonistic polypeptide.

IN-60:

MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVV PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGP TTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDEC(S-maleimide-PE G12-human insulin) (S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO:144),

the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with α-amino group at the N-terminus of the B chain of the human insulin, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at the C-terminus of the interleukin-1 receptor antagonistic protein.

IN-61:

MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVV PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGP TTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDEC(S-maleimide-PE G12-human insulin desB30T)(S is the sulphur atom on the side chain of a cysteine residue) (SEQ ID NO:145),

the N-hydroxylsuccinimide of the linking group, i.e. maleimide-PEG12-NHS, is reacted with the ε-amino group on the side chain of a lysine in the human insulin desB30T B29, and the maleimide is reacted with the mercapto group on the side chain of a cysteine at the C-terminus of the interleukin-1 receptor antagonistic polypeptide.

In the sequences of this section, [1] to [6] indicate the Nos. of the cysteines; 3 pairs of disulfide bonds are formed by six cysteines in the compound, in which the A chain and the B chain are connected by two pairs of interchain disulfide bonds, a pair of intrachain disulfide bond is present in the A chain, and the specific positions for the three pairs of the disulfide bonds are: C_([1]) and C_([4]) forming a disulfide bond, C_([2]) and C_([6]) forming a disulfide bond, and C_([3]) and C_([5]) forming a disulfide bond.

The structure of the reagent of maleimide-PEG12-NHS is shown as follows:

The structure of the reagent after reacted with the interleukin-1 receptor antagonistic protein and the insulin receptor binding polypeptide is shown as follows:

3. The fusion protein consisting of the GIP receptor binding polypeptide and the interleukin-1 receptor antagonistic protein

In one embodiment, the sequence of the GIP receptor binding polypeptide is

(X_(GI1)X_(GI2)X_(GI3)GT)_(t0)X_(GI6)X_(GI7)SDX_(GI12)X_(GI13)X_(GI14)DX_(GI16)X_(GI17)X_(GI18)QX_(GI20)X_(GI21)F X_(GI23)X_(GI24)WLX_(GI27)X_(GI28)X_(GI29)X_(GI30)X_(GI31)X_(GI32)X_(GI33)X_(GI34)X_(GI35)X_(GI36)X_(GI37)X_(GI38)X_(GI39)X_(GI40)X_(GI41)X_(GI42)X_(GI43) (SEQ ID NO:439), wherein,

X_(GI1) is a tyrosine, N-acetyl tyrosine, pyroglutamyl-tyrosine, glucitol-tyrosine, D-histidine, α, α-2 methylimidazolyl acetic acid (DMIA), N-methylhistidine, α-methylhistidine, imidazole acetic acid, deamino-histidine, hydroxylhistidine, acetylhistidine, homohistidine, N-palmitic acid-tyrosine, N-Fmoc-tyrosine, or absent; X_(GI2) is an alanine, D-alanine, D-serine, serine, valine, glycine, N-methylserine, N-methylalanine, 2-methylalanine, or absent; X_(GI3) is a glutamic acid, hydroxyl proline, proline, or absent; X_(GI6) is a phenylalanine, or absent; X_(GI7) is an isoleucine, or threonine; X_(GI10) is a tyrosine, tryptophan, phenylalanine, valine, lysine, ornithine, glutamic acid, leucine, general formula 1, or general formula 2; X_(GI12) is an isoleucine, serine, lysine, arginine, general formula 1, or general formula 2; X_(GI13) is an alanine, glutamine, or tyrosine; X_(GI14) is a methionine or leucine; X_(GI16) is a lysine, arginine, serine, glutamic acid, glutamine, homoglutamic acid, homocysteine, threonine, glycine, 2-methylalanine, general formula 1, or general formula 2; X_(GI17) is an isoleucine, glutamine, glutamic acid, arginine, general formula 1, or general formula 2; X_(GI18) is a histidine, alanine, arginine, serine, threonine, or glycine; X_(GI20) is a glutamine, lysine, alanine, serine, threonine, citrulline, arginine, ornithine, 2-methylalanine, other α,α-disubstituted amino acids, general formula 1, or general formula 2; X_(GI21) is an aspartic acid, glutamic acid, homoglutamic acid, homocysteine, leucine, general formula 1, or general formula 2; X_(GI123) is a valine, or isoleucine; X_(GI24) is a glutamine, glutamic acid, asparagine, alanine, serine, threonine, 2-methylalanine, general formula 1, or general formula 2; X_(GI27) is a leucine, isoleucine, norleucine, lysine, valine, methionine, general formula 1, or general formula 2; X_(GI28) is a glycine, alanine, lysine, asparagine, general formula 1, or general formula 2; X_(GI29) is a glutamine, glycine, threonine, alanine, general formula 1, or general formula 2; X_(GI30) is a lysine, arginine, glycine, general formula 1, or general formula 2; X_(GI31) is a —NH₂, glycine, proline, or absent; X_(GI32) is a lysine, arginine, serine, general formula 1, general formula 2, or absent; X_(GI33) is a lysine, arginine, serine, general formula 1, general formula 2, or absent; X_(GI34) is an asparagine, glycine, or absent; X_(GI35) is an asparagine, alanine, or absent; X_(GI36) is a tryptophan, proline, or absent; X_(GI37) is a lysine, arginine, proline, general formula 1, general formula 2, or absent; X_(GI38) is a histidine, proline, or absent; X_(GI39) is an asparagine, serine, or absent; X_(GI40) is an isoleucine,-NH₂, general formula 1, general formula 2, or absent; X_(GI41) is a threonine or absent; X_(GI42) is a glutamine or absent; X_(GI43) is a lysine, cysteine, PSSGAPPPS (SEQ ID NO:438), general formula 3, general formula 4, or absent; t0 is 0 or 1.

In one embodiment, the sequence from positions 31-42 in the GIP can be deleted by 1-12 of amino acid residues starting from the C-terminus. In one embodiment, the sequence from positions 31-42 in the GIP can be substituted with PSSGAPPPS (SEQ ID NO:438), GPSSGAPPPS (SEQ ID NO:437), X_(GI)PSSGAPPPS (SEQ ID NO:440), PSSGAPPPX_(GI)-NH₂ (SEQ ID NO:441), PSSGAPPPSX_(GI)-NH₂ (SEQ ID NO:442), in which X_(GI) is general formula 1 or general formula 2.

The GIP receptor binding polypeptide and the interleukin-1 receptor antagonistic protein are able to protect pancreas islet β-cells by similar or distinct mechanisms, thereby treating diabetes mellitus. Accordingly, the fusion protein, dimeric protein or cross-linking protein formed by joining the two compounds may attain a therapeutic effect more excellent than that with either of the compounds alone, due to the synergistic effect by the two compounds. Furthermore, one of the tissues in which the GIP receptors are mainly distributed is the pancreatic islet. The GIP receptor binding polypeptide can serve as a target for the fusion protein, dimeric protein or cross-linking protein and enrich the interleukin-1 receptor antagonistic protein within and around the pancreatic islet, more effectively exerting actions such as anti-inflammation, etc.

The GIP receptor binding polypeptide is linked to the interleukin-1 receptor antagonistic protein in various linkages through an alternative linking group (or spacer group).

(1) The GIP receptor binding polypeptide - linking group or spacer group-the interleukin-1 receptor antagonistic protein

In one embodiment, the GIP receptor binding polypeptide and the interleukin-1 receptor antagonistic protein form a single chain compound via a linking group, for which the structure is (from left to right corresponding to the amino acid sequence from N-terminus to C-terminus):

GIP receptor binding polypeptide - linking group (or spacer group)-interleukin-1 receptor antagonistic protein; or

interleukin-1 receptor antagonistic protein-linking group (or spacer group)-GIP-1 receptor binding polypeptide.

In one embodiment, the sequence of the fusion protein is

(SEQ ID NO: 443) U_(L)-(X_(GI1)X_(GI2)X_(GI3)GT)_(t0)X_(GI6)X_(GI7)SDX_(GI10)SX_(GI12)X_(GI13) X_(GI14)DX_(GI16)X_(GI17)X_(GI18)QX_(GI20)X_(GI21)FX_(GI23)X_(GI24)WIX_(GI27) X_(GI28)X_(GI29)X_(GI30)X_(GI31)X_(GI32)X_(GI33)X_(GI34)X_(GI35)X_(GI36) X_(GI37)X_(GI38)X_(GI39)X_(GI40)X_(GI41)X_(GI42)X_(GI43)-L_(j)-X_(IL0) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNL EEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGD ETRLQLEAVX_(IL84)ITDLSENRKQDKRFAFIRSDSGPTTSFES AAX_(IL116)PGWFLX_(IL122)TAMEADQPVSLTNMPDEGV MVTKFYFQEDE; or (SEQ ID NO: 444) U_(L)-(X_(GI1)X_(GI2)X_(GI3)GT)_(t0)X_(GI6)ISDX_(GI10)SX_(GI12)AMDX_(GI16) IHQQDFVNWLX_(GI27)X_(GI28)QX_(GI30)(GX_(GI32)X_(GI33)NDWX_(GI37) HNITQ)_(t1)(PSSGAPPPS)_(t2)-(GGGGS)_(m)-X_(L)-(GGGGS)_(n)- RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKID VVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVX_(IL84)ITD LSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFLCTA MEADQPVSLTNMPDEGVMVTKFYFQEDE; or (SEQ ID NO: 445) U_(L)-X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDKIHQQDFV NWLLAQK(GKKNDWKHNITQ)_(t1)(PSSGAPPPS)_(t2)-(GGGGS)_(m)- X_(L)-(GGGGS)_(n)-RPSGRKSSKMQAFRIWDVNQKTFYLRNNQ LVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGICIVICLSCVKSGDET RLQLEAVX_(IL84)ITDLSENRKQDKRFAFIRSDSGPTTSFESAA X_(IL116)PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE; or (SEQ ID NO: 446) U_(L)-X_(GI6)ISDYSIAMDKIHQQDFVNWLLAQK(GKKNDWKHNITQ)_(t1)(PS SGAPPPS)_(t2)-(GGGGS)_(m)-X_(L)-(GGGGS)_(n)-RPSGRKSSKMQAF RIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLG IHGGKMCLSCVKSGDETRLQLEAVX_(IL84)ITDLSENRKQDKRFAFIRSD SGPTTSFESAAX_(IL116)PGWFLCTAMEADQPVSLTNMPDEGVMVTKF YFQEDE,

in the above-mentioned sequences, m and n are each 0, 1, 2, 3, 4, 5, or 6; t0, t1, and t2 are each 0 or 1, and other variables are defined as herein.

In one embodiment, the GIP receptor binding polypeptide, the interleukin-1 receptor antagonistic protein, and a biomacromolecule form a single chain compound, for which the structure is (from left to right corresponding to the amino acid sequence from N-terminus to C-terminus):

GIP receptor binding polypeptide—linking group (or spacer group)-biomacromolecule-linking group (or spacer group)-interleukin-1 receptor antagonistic protein;

GIP receptor binding polypeptide—linking group (or spacer group)-interleukin-1 receptor antagonistic protein-linking group (or spacer group)-biomacromolecule; in which the biomacromolecule may be an albumin or IgG Fc, etc.

In one embodiment, the sequence of the fusion protein comprising the human albumin is:

(X_(GI1)X_(GI2)X_(GI3)GT)_(t0)X_(GI6)ISDYSIAMDKIHQQDFVNWLLAQK(GKKNDWKHNITQ)_(t1)(PSSGAPPPS)_(t2)-(GGGGS)_(m)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFE DHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCA KQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYF YAPELLFFA KRYKAAFTECCQAAD KAACLLPKLDELRDEGKASSAKQRLKCASLQK FGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLA KYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFD EFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCF SALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLK AVMDDFAAFVEKCCKADDKETCFAEEGKKLVAAS QAALGL-(GGGGS)_(n)-RPSGRKSS KMQAPRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGK MCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLC TAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:447), in which m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as herein.

In one embodiment, the sequence of the fusion protein comprising the human albumin is:

(X_(GI1)X_(GI2)X_(GI3)GT)_(t0)X_(GI6)ISDYSIAMDKIHQQDFVNWLLAQK(GKKNDWKHNITQ)_(t1)(PSSGAPPPS)_(t2)-(GGGGS)_(m)-RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQ GPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQ DKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQED E-(GGGGS)_(n)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTE FAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQ HKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRY KAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGM FLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQN LIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEA KRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVP KEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVE KCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO:448),wherein m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as herein. In one embodiment, the sequence of the fusion protein comprising the human albumin is:

(X_(GI1)X_(GI2)X_(GI3)GT)_(t0)X_(GI6)OSDYSIAMDKIHQQDFVNWLLAQK(GKKNDWKHNITQ)_(t1)(PSSGAPPPS)_(t2)(X_(GI1) X_(GI2)X_(GI3)GT)_(t0)X_(GI6)ISDYSIAMDKIHQQDFVNWLLAQK(GKKND WKHNITQ)_(t1)(PSSGAPPPS)_(t2)-(GGGGS)_(m)-RPSGRKSSKMQAFRIWDVNQKTFYLRNNQL VAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITD LSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVT KFYFQEDE-(GGGGS)_(n)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVK LVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPE RNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPEL LFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERA FKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICE NQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAK DVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKP LVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSK CCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALE VDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMD DFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO:449);

In one embodiment, the sequence of the fusion protein comprising the human albumin is:

(X_(GI1)X_(GI2)X_(GI3)GT)_(t0)X_(GI6)ISDYSIAMDKIHQQDFVNWLLAQK(GKKNDWKHNITQ)_(t1)(PSSGAPPPS)_(t2)(X_(GI1)X_(GI2)X_(GI3)GT)_(t0)X_(GI6)ISDYSIAMDKIHQQDFVNWLLAQK(GKKND WKHNITQ)_(t1)(PSSGAPPPS)_(t2)-(GGGGS)_(m)-DAHKSEVAHRFKDLGEENFKALVLIAFAQY LQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGE MADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEI ARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRL KCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECA DDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVES KDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPH ECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLV EVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPK ATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL-(GGGGS)_(n)-RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALF LGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAA CPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:450); wherein m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as herein.

In one embodiment, the sequence of the fusion protein comprising IgG1 Fc is:

(X_(GI1)X_(GI2)X_(GI3)GT)_(t0)X_(GI6)ISDYSIAMDKIHQQDFVNWLLAQK(GKKNDWKHNITQ)_(t1)(PSSGAPPPS)_(t2)-(GGGGS)_(m)-RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQ GPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQ DKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQED E-(GGGGS)_(n)-AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO:451), wherein m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as herein.

In one embodiment, the sequence of the fusion protein comprising IgG1 Fc is:

(X_(GI1)X_(GI2)X_(GI3)GT)_(t0)X_(GI6)ISDYSIAMDKIHQQDFVNWLLAQK(GKKNDWKHNITQ)_(t1)(PSSGAPPPS)_(t2)-(GGGGS)_(m)-AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGIC-(GGGGS)_(n)-RPSGRKSSKMQAFRIWDVNQKT FYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQ LEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNM PDEGVMVTKFYFQEDE (SEQ ID NO:452); wherein m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as herein.

All of the fusion proteins comprising IgG1 Fc are dimeric proteins connected by an interchain disulfide bond formed between the cysteines of the Fc portions of the two monomeric proteins.

In one embodiment, the sequence of the fusion protein comprising IgG4 Fc and the interleukin-1 receptor antagonistic protein is (X_(GI1)X_(GI2)X_(GI3)GT)_(t0)X_(GI6)ISDYSIAMDKIHQQDFVNWLLAQK(GKKNDWKHNITQ)_(t1)(PSS GAPPPS)_(t2)-(GGGGS)_(m)-RPSGRKSSKMQAPRIWDVNQKTFYLRNNQLVAGYLQGPNVN LEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFA FIRSDSGPTTSFESAAX_(IL116)PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE-(GGGGS)_(n)-AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC_(a)VVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC_(a)K VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC_(b)LVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC_(b)SVMHEALHNH YTQKSLSLSLG (SEQ ID NO:453), wherein m and n are each 0, 1, 2, 3, 4, 5, or 6 and other variables are defined as herein.

The dimeric protein is a homodimer consisting of two identical sequences mentioned above. Each cysteine C underlined and the cysteine at the corresponding position in the other monomer form an interchain disulfide bond; an intrachain disulfide bond is formed between the C_(a)s in each monomer; and an intrachain disulfide bond is formed between the C_(b)s in each monomer.

In this section, the fusion protein comprising the GIP receptor binding polypeptide and the interleukin-1 receptor antagonistic protein is selected from GI-1:

(SEQ ID NO: 155) YSEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSRP SGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYF QEDE; GI-2: (SEQ ID NO: 156) Ac-YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQRPSG RKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEP HALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIR SDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQ EDE; GI-3: (SEQ ID NO: 157) Ac-YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGG SRPSGRKSSKMQAPRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDV VPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKR FAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKF YFQEDE; GI-4: (SEQ ID NO: 158) Ac-YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGG SGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLE EKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENR KQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGV MVTKFYFQEDE; GI-5: (SEQ ID NO: 159) Ac-YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGG SGGGGSGGGGSRPSGRKSSKMQAPRIWDVNQKTFYLRNNQLVAGYLQGP NVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITD LSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNM PDEGVMVTKFYFQEDE; GI-6: (SEQ ID NO: 160) YAPGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQRPSGRKSS KMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFL GIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPT TSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE; GI-7: (SEQ ID NO: 161) YAPGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSRPS GRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEP HALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRS DSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE; GI-8: (SEQ ID NO: 162) YAPGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGG GGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKI DVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQD KRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVT KFYFQEDE; GI-9: (SEQ ID NO: 163) YAPGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGG GGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVN LEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSE NRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDE GVMVTKFYFQEDE; GI-10: (SEQ ID NO: 164) ISDYSIAMDKIHQQDFVNWLLAQKPSSGAPPPSGGGGSGGGGSRPSGRKS SKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALF LGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGP TTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE; GI-11: (SEQ ID NO: 165) YGEGTFISDYSIAMDKIHQQDFVNWLLAQKPSSGAPPPSGGGGSGGGGSG GGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKI DVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDK RFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKF YFQEDE; GI-12: (SEQ ID NO: 166) YGEGTFISDYSIAMDKIHQQDFVNWLLAQKPSSGAPPPSGGGGSRPSGRK SSKMQAPRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHAL FLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSG PTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE; GI-13: (SEQ ID NO: 167) Ac-YAEGTFISDYSIAMDKIHQQDFVNWLLAQKPSSGAPPPSGGGGSGG GGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVN LEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSE NRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDE GVMVTKFYFQEDE; GI-14: (SEQ ID NO: 168) Ac-YAEGTFISDYSIAMDKIHQQDFVNWLLAQKPSSGAPPPSGGGGSGG GGSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKI DVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQD KRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVT KFYFQEDE; GI-15: (SEQ ID NO: 169) Ac-YAEGTFISDYSIAMDKIHQQDFVNWLLAQKPSSGAPPPSGGGGSRP SGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFY FQEDE; GI-16: (SEQ ID NO: 170) FISDYSIAMDKIHQQDFVNWLLAQKGGGGSGGGGSRPSGRKSSKMQAFR IWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGG KMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFE SAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE; GI-17: (SEQ ID NO: 171) FISDYSIAMDKIHQQDFVNWLLAQKGGGGSRPSGRKSSKMQAFRIWDVN QKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLS CVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACP GWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE; GI-18: (SEQ ID NO: 172) FISDYSIAMDKIHQQDFVNWLLAQKPSSGAPPPSGGGGSGGGGSRPSGRK SSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHA LFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSD SGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE; GI-19: (SEQ ID NO: 173) YAPGTFISDYSIAMDKIHQQDFVNWLLAQKRPSGRKSSKMQAFRIWDVN QKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLS CVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACP GWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE; GI-20: (SEQ ID NO: 174) YAPGTFISDYSIAMDKIHQQDFVNWLLAQKPSSGAPPPSGGGGSRPSGR KSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPH ALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRS DSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE; GI-21: (SEQ ID NO: 175) YAPGTFISDYSIAMDKIHQQDFVNWLLAQKPSSGAPPPSGGGGSGGGGSR PSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFI RSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQ EDE; GI-22: (SEQ ID NO: 176) Ac-YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGG SRPSGRKSSKMQAPRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVV PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFA FIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQ EDEGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKL VNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCA KQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEI ARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKAS SAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAE VENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYS VVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNC ELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPE AKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALE VDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL; GI-23: (SEQ ID NO: 177) YAPGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSRPS GRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIE PHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFI RSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQED EGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVN EVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQ EPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA KQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTE CCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVE NDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVV LLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCEL FEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAK RMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVD ETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQL KAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL;

(2) A dimeric protein/cross-linking protein of the interleukin-1 receptor antagonistic protein—the linking or spacer group—the GIP receptor binding polypeptide

In such compounds, the linking group (or the spacer group) is linked at a non-terminal position of the interleukin-1 receptor antagonistic protein and of the GIP receptor binding polypeptide.

In one embodiment, the sequence of the fusion protein is U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIE PHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTS FESAAC (linking group—GLP receptor binding polypeptide) PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:454); each of the variables is defined as herein.

In another embodiment, the sequence of the fusion protein is U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIE PHALFLGIHGGKMCLSCVKSGDETRLQLEAVC(linking group—GIP receptor binding polypeptide)

ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGV MVTKFYFQEDE (SEQ ID NO:455); each of the variables is defined as herein.

In one particular embodiment, the molecular structure of the linking group may be maleimide-PEG-maleimide or I—CH₂—CONH-PEG-NHCO—CH₂—I; in one particular embodiment, the molecular structure of the linking group may be maleimide-(CH₂)_(n)-maleimide or I—CH₂—CONH—(CH₂)_(n)—NHCO—CH₂—I, in which n may be an integer from 1 to 30; in one particular embodiment, the molecular structure of the linking group may be maleimide -PEG-NHS. The GIP receptor binding polypeptide may typically react with the linking group through the amino group on the side chain of a lysine or the mercapto group on the side chain of a cysteine.

In one embodiment, the sequence of the GIP receptor binding polypeptide may be U_(L)-(X_(GI1)X_(GI2)X_(GI3)GT)_(t0)X_(GI6)ISDX_(GI10)SX_(GI12)AMDX_(GI16)IHQQDFVNWLX_(GI27)X_(GI28)QX_(GI30)(GX_(GI32)X_(GI33)NDWX_(GI37)HNITQ)_(t1)(PSSGAPPPS)_(t2) (SEQ ID NO:456), wherein each of the variables is defined as herein.

In one particular embodiment, the sequence of the GIP receptor binding polypeptide may be one of the following sequences:

(SEQ ID NO: 457) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWK HNITQC; (SEQ ID NO: 458) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWC HNITQ; (SEQ ID NO: 459) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDKIHQQDFVNWLLAQKGKCNDWK HNITQ; (SEQ ID NO: 460) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDKIHQQDFVNWLLAQKGCKNDWK HNITQ; (SEQ ID NO: 461) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDKIHQQDFVNWLLAQCGKKNDWK HNITQ; (SEQ ID NO: 462) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDCIHQQDFVNWLLAQKGKKNDWK HNITQ; (SEQ ID NO: 463) X_(GI1)X_(GI2)X_(GI3)GTFISDYSCAMDKIHQQDFVNWLLAQKGKKNDW KHNITQ; (SEQ ID NO: 464) X_(GI1)X_(GI2)X_(GI3)GTFISDCSIAMDKIHQQDFVNWLLAQKGKKNDWK HNITQ; (SEQ ID NO: 465) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDKIHQQDFVNWLLAQKC; (SEQ ID NO: 466) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDCIHQQDFVNWLLAQK-NH₂; (SEQ ID NO: 467) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDKIHQQDFVNWLLAQKPSSGAPPPSC; (SEQ ID NO: 468) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDKIHQQDFVNWLLAQKPSSGAPPPC- NH₂; (SEQ ID NO: 469) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDKIHQQDFVNWLLAQCPSSGAPPPS- NH₂; (SEQ ID NO: 470) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDCIHQQDFVNWLLAQKPSSGAPPPS- NH₂; (SEQ ID NO: 471) X_(GI1)X_(GI2)X_(GI3)GTFISDYSCAMDKIHQQDFVNWLLAQKPSSGAPPPS- NH₂; (SEQ ID NO: 472) X_(GI1)X_(GI2)X_(GI3)GTFISDCSIAMDKIHQQDFVNWLLAQKPSSGAPPPS- NH₂; (SEQ ID NO: 473) FISDYSIAMDKIHQQDFVNWLLAQKC; (SEQ ID NO: 474) FISDYSIAMDKIHQQDFVNWLLAQC-NH₂; (SEQ ID NO: 475) FISDYSIAMDCIHQQDFVNWLLAQK-NH₂; (SEQ ID NO: 476) ISDYSIAMDKIHQQDFVNWLLAQC-NH2; or (SEQ ID NO: 477) ISDYSIAMDCIHQQDFVNWLLAQK-NH₂;

each of the variables is defined as herein;

The GIP-1 receptor binding polypeptide is linked to the interleukin-1 receptor antagonistic protein by reacting the mercapto group on the side chain of a cysteine with the maleimide or iodoacetyl on the linking group.

In one particular embodiment, the sequence of the GIP receptor binding polypeptide is one of the following sequences:

(SEQ ID NO: 478) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDRIHQQDFVNWLLAQRGRRNDWR HNITQK; (SEQ ID NO: 479) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDRIHQQDFVNWLLAQRGRRNDWK HNITQ; (SEQ ID NO: 480) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDRIHQQDFVNWLLAQRGRKNDWR HNITQ; (SEQ ID NO: 481) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDRIHQQDFVNWLLAQRGKRNDWR HNITQ; (SEQ ID NO: 482) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDRIHQQDFVNWLLAQKGRRNDWR HNITQ; (SEQ ID NO: 483) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDKIHQQDFVNWLLAQRGRRNDWR HNITQ; (SEQ ID NO: 484) X_(GI1)X_(GI2)X_(GI3)GTFISDYSKAMDRIHQQDFVNWLLAQRGRRNDWR HNITQ; (SEQ ID NO: 485) X_(GI1)X_(GI2)X_(GI3)GTFISDKSIAMDRIHQQDFVNWLLAQRGRRNDWR HNITQ; (SEQ ID NO: 486) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDRIHQQDFVNWLLAQRPSSGAPPPSK; (SEQ ID NO: 487) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDRIHQQDFVNWLLAQRPSSGAPPPK- NH₂; (SEQ ID NO: 488) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDRIHQQDFVNWLLAQKPSSGAPPPS- NH₂; (SEQ ID NO: 489) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDKIHQQDFVNWLLAQRPSSGAPPPS- NH₂; (SEQ ID NO: 490) X_(GI1)X_(GI2)X_(GI3)GTFISDKSIAMDRIHQQDFVNWLLAQRPSSGAPPPS- NH₂; (SEQ ID NO: 491) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDRIHQQDFVNWLLAQK-NH₂; (SEQ ID NO: 492) X_(GI1)X_(GI2)X_(GI3)GTFISDYSIAMDKIHQQDFVNWLLAQR-NH₂; (SEQ ID NO: 493) FISDYSIAMDRIHQQDFVNWLLAQK-NH₂; (SEQ ID NO: 494) FISDYSIAMDKIHQQDFVNWLLAQR-NH₂; (SEQ ID NO: 495) ISDYSIAMDRIHQQDFVNWLLAQK-NH₂; or SEQ ID NO: 496) ISDYSIAMDKIHQQDFVNWLLAQR-NH₂;

each of the variables is defined as herein;

The GIP receptor binding polypeptide is reacted with the N-hydroxyl succinimidyl ester of the linking group through the amino group on the side chain of a lysine in the polypeptide, thus linking to the interleukin-1 receptor antagonistic protein.

In one embodiment, the sequence of the fusion protein is

(GIP-1 receptor binding polypeptide—linking group)-C_(IL0)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKI DVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVX_(IL84)ITDLSENRKQDKRFAFIR SDSGPTTSFESAAX_(IL116)PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:497), each of the variables being defined as herein.

In one embodiment, the linking group has an aldehyde group and is linked to the the N-terminus of interleukin-1 receptor antagonistic protein through reductive amination; in another embodiment, C_(IL0) cysteine is linked to the linking group via the mercapto group on the side chain, and then is linked to the GLP receptor binding polypeptide.

In one embodiment, the sequence of the fusion protein is U_(L)-X_(IL0)RPSGRKSSKMQAPRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIE PHALFLGIHGGKMCLSCVKSGDETRLQLEAVX_(IL84)ITDLSENRKQDKRFAFIRSDSGPT TSFESAAX_(IL116)PGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDEC_(IL153)(linking group—GLP receptor binding polypeptide) (SEQ ID NO:498), each of the variables being defined as herein.

The GIP receptor binding polypeptide may typically react with the linking group through the amino or mercapto group on the side chain of an amino acid residue.

In one embodiment, the insulin receptor binding polypeptide is a modified conjugate.

The sequences of the dimeric protein/cross-linking protein of the GIP receptor binding polypeptide and the interleukin-1 receptor antagonistic protein are selected from:

GI-24:

MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVV PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC (S-maleimide-PEG11-maleimide-S-GIP receptor binding polypeptide)ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLT NM PDEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:178); wherein the GIP receptor binding polypeptide has the following sequence:

Ac-YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWCHNITQ (SEQ ID NO: 197) and is linked to the interleukin-1 receptor binding protein by reacting the mercapto group on the side chain of a cysteine with the maleimide;

GI-25:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTT SFESAAC(S-maleimide-PEG40K-maleimide-S-GIP receptor binding polypeptide)PGWFLCTAMEADQPVSLT NMPDEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:179); wherein the GIP receptor binding polypeptide has the following sequence:

Ac-YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQC (SEQ ID NO: 198) and is linked to the interleukin-1 receptor binding protein by reacting the mercapto group on the side chain of a C-terminal cysteine with the maleimide;

GI-26:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTT SFESAAC(S-maleimide-PEG40K-maleimide-S-GIP receptor binding polypeptide)PGWFLCTAMEADQPVSLT NMPDEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:180); wherein the GIP receptor binding polypeptide has the following sequence:

YAPGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQC (SEQ ID NO: 199) and is linked to the interleukin-1 receptor binding protein by reacting the mercapto group on the side chain of a C-terminal cysteine with the maleimide;

GI-27:

MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVV PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGP TTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDEC(S-maleimide-PEG11-maleimide-S-GIP receptor binding polypeptide)(S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:181); wherein the sequence of the GIP receptor binding polypeptide is YAibEGTFISDYSIAMDKIHQQDFVNWLLAQC-NH₂ (SEQ ID NO:200);

GI-28:

C(S-maleimide-PEG11-maleimide-S-GIP receptor binding polypeptide)RPSGRKS SKMQA PRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSC VKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAC[S—CH₂—CONH—(CH₂CH₂O)₄—(CH₂)₂—NH—(N^(α)—(HOOC(CH₂)₁₆CO)-γ-Glu)]PGWFLCTAMEADQPVSLTNMPD EGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:182); wherein the GIP receptor binding polypeptide has the sequence of YAibEGTFISDYSIAMDKIHQQDFVNWLLAQKPSSGAPPPSC(SEQ ID NO:201) and is linked to the interleukin-1 receptor binding protein by reacting the mercapto group on the side chain of a C-terminal cysteine with the maleimide;

GI-29:

MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVV PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVC(S-maleimide-PEG40K-maleimide-S-GIP receptor binding polypeptide)ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLT N-MPDEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:183); wherein the GIP receptor binding polypeptide has the sequence of Ac-YAEGTFISDYSIAMDKIHQQDFVNWLLAQKPS SGAPPPC-NH₂(SEQ ID NO:202) and is linked to the interleukin-1 receptor binding protein by reacting the mercapto group on the side chain of a C-terminal cysteine with the maleimide;

GI-30:

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTT SFESAAC(S-maleimide-PEG11-maleimide-S-GIP receptor binding polypeptide)PGWFLCTAMEADQPVSL TNMPDEGVMVTKFYFQEDE(S is the sulphur atom on the side chain of a cysteine residue)(SEQ ID NO:184); wherein the GIP receptor binding polypeptide has the sequence of FISDYSIAMDKIHQQDFVNWLLAQKC-NH₂(SEQ ID NO:203) and is linked to the interleukin-1 receptor binding protein by reacting the mercapto group on the side chain of a C-terminal cysteine with the maleimide.

Pharmaceutical Compositions and the Use

In a further aspect of the invention, a pharmaceutical composition comprising a therapeutically effective amount of a fusion protein or a fusion protein conjugate according to the present invention and a pharmaceutically acceptable carrier or additive, optionally other anti diabetic compounds, is provided for the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycemia. A fusion protein according to the present invention can be used for the manufacture of a pharmaceutical composition for use in the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycemia.

Injectable compositions of the fusion protein of the present invention can be prepared using the conventional techniques of the pharmaceutical industry which involve dissolving and mixing the suitable ingredients to give the final desired product. Thus, according to one procedure, a fusion protein according to the present invention is dissolved in an amount of water, of which the volume is somewhat less than the final volume of the composition to be prepared. An isotonic agent, a preservative and a buffer are added as required and the pH value of the solution is adjusted, if necessary, using an acid, e.g. hydrochloric acid, or a base, e.g. sodium hydroxide. Finally, the volume of the solution is adjusted with water to give the desired concentration.

In a further embodiment of the present invention, the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethane, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each one of these specific buffers constitutes an alternative embodiment of the invention.

In a further embodiment of the present invention, the preservative may be selected from the group consisting of phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate,propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, 2-phenoxyethanol, benzyl alcohol, chlorobutanol, thiomersal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, benzethonium chloride, chlorphenesine or mixtures thereof. The preservative is present in a concentration from 0.1 mg/ml to 20 mg/ml, preferably from 0.1 mg/ml to 5 mg/ml. In a further embodiment of the invention, the preservative is present in a concentration from 5 mg/ml to 10 mg/ml. Each one of these specific preservatives constitutes an alternative embodiment of the invention. The use of a preservative in pharmaceutical compositions is well-known to the skilled person. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19^(th) edition, 1995.

In a further embodiment of the present invention, the formulation further comprises an isotonic agent which may be selected from the group consisting of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an amino acid, an alditol (e.g. glycerol, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol), polyethyleneglycol (e.g. PEG400), or mixtures thereof. Any sugar such as mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na may be used. In one embodiment, the sugar additive is sucrose. Sugar alcohol is defined as a C4-C8 hydrocarbon having at least one —OH group and includes, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In one embodiment, the sugar alcohol additive is mannitol. The sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, as long as the sugar or sugar alcohol is soluble in the liquid preparation and does not adversely effect the stabilizing effects achieved using the methods of the invention. In one embodiment, the sugar or sugar alcohol concentration is between 1 mg/ml and 150 mg/ml. In a further embodiment of the invention, the isotonic agent is present in a concentration from 1 mg/ml to 50 mg/ml. In a further embodiment of the invention, the isotonic agent is present in a concentration from 1 mg/ml to 7 mg/ml. In a further embodiment of the invention, the isotonic agent is present in a concentration from 8 mg/ml to 24 mg/ml. In a further embodiment of the invention, the isotonic agent is present in a concentration from 25 mg/ml to 50 mg/ml. Each one of these specific isotonic agents constitutes an alternative embodiment of the invention. The use of an isotonic agent in pharmaceutical compositions is well-known to the skilled person. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19^(th) edition, 1995.

Typical isotonic agents are sodium chloride, mannitol, dimethyl sulfoxide and glycerol, and typical preservatives are phenol, m-cresol, methyl p-hydroxybenzoate and benzyl alcohol.Examples of surfactants are sodium acetate, glycylglycine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and sodium phosphate.

EXAMPLES

Protective Groups

Acm acetamidomethy; Alloc or AOC allyloxycarbonyl; Bom, benzyloxymethyl; Br-Z, 2-bromobenzyloxycarbonyl; tBu, t-butyl; Bz, benzoyl; Bzl, benzyl; Boc:Butyloxy carbonyl; CHO formyl; cHx, cyclohexyl; Cbz or Z benzyloxycarbonyl; Cl-Z, 2-chlorobenzyloxycarbonyl; Fm, 9-fluorenylmethyl; Fmoc, 9-fluorenylmethoxycarbonyl; Mtt, 4-methyltrityl; Npys, 3-nitro-2-pyridines ulfenyl; Pmc, (2,2,5,7,8-pentamethylchroman-6-sulphonyl; Tos,4-toluenesulphonyl; Trt, triphenylmethyl; Xan, xanthyl.

Reagents and Solvents

ACN, acetonitrile; BOP, benzotriazol-1-yloxytris(dimethylamino) phosphonium hexafluorophosphate; DCC, N,N′-Dicyclohexylcarbodiimide; DCM, dichloromethane; DEPBT, 3-(Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one; DIC, N,N′-Diisopropylcarbodiimide; DIPEA (or DIEA), diisopropylethylamine; DMAP, 4-N,N-dimethylaminopyridine; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EDC or EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.HCl; EtOAc,ethyl acetate; HBTU, O-(1H-benzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; HOBT, 1-hydroxybenzotriazole; Cl-HOBT 6-chloro-1-hydroxybenzotriazole; NMM, N-Methylmorpholine; NMP, N-methylpyrrolidinone; Piperidine, Su, succinimide; TEA, triethylamine; TFA, trifluoroacetic acid; TFE 2,2,2-Trifluoroethanol; THF, tetrahydrofuran; TIS, triisopropylsilane.

Reagents used in the present invention are purchasable from an ordinary shop for chemical reagents or bio-reagent/preparations or reagents conventionally formulated in the field, unless otherwise specified. All of the experiments and the steps thereof can be performed by the person skilled in the art according to the disclosure in the present invention and the conventional techniques in the field.

Interleukin-1 Receptor Antagonistic Protein Open Reading Frame Sequence:

(SEQ ID NO: 500) ATGGAAATCTGCAGAGGCCTCCGCAGTCACCTAATCACTCTCCTCCTCTT CCTGTTCCATTCAGAGACGATCTGCCGACCCTCTGGGAGAAAATCCAGCA AGATGCAAGCCTTCAGAATCTGGGATGTTAACCAGAAGACCTTCTATCTG AGGAACAACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTT AGAAGAAAAGATAGATGTGGTACCCATTGAGCCTCATGCTCTGTTCTTGG GAATCCATGGAGGGAAGATGTGCCTGTCCTGTGTCAAGTCTGGTGATGAG ACCAGACTCCAGCTGGAGGCAGTTAACATCACTGACCTGAGCGAGAACAG AAAGCAGGACAAGCGCTTCGCCTTCATCCGCTCAGACAGTGGCCCCACCA CCAGTTTTGAGTCTGCCGCCTGCCCCGGTTGGTTCCTCTGCACAGCGATG GAAGCTGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCAT GGTCACCAAATTCTACTTCCAGGAGGACGAGTAG.

IL-1ra amino acid sequence:

(SEQ ID NO: 501) MEICRGLRSHLITLLLFLFHSETICRPSGRKSSKMQAFRIWDVNQKTFYL RNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDE TRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAM EADQPVSLTNMPDEGVMVTKFYFQEDE*.

1. Cloning of cDNA of interleukin-1 receptor antagonistic proteinand construction of expression vector

1.1 Designing the primers containing restriction enzyme digestion sites for insertion of PCR amplification product of DNA

The cDNA sequence of IL-1Ra was obtained from human hepatic tissue using RT-PCR technology. Firstly, we need to check the sequence of cDNA to be amplified to identify that these enzyme digestion sites are not in the cDNA, and design the appropriate primers to allow specific amplification of the 5′ end of the cDNA. The cDNA sequence encoding complete or mature IL-1Ra was firstly amplified, and then ligated to pSUMO vector after digestion by restriction enzyme. Many pSUMO vectors are commercially available, such as the products of Lifesensors, Invitrogen, can be used for the proteins in the present invention.

Primer Design

Forward primer 1: (5′ GGCGGTCTCTAGGT-ATGGAAATCTGCAGA-3′) (SEQ ID NO:502) for cloning the complete sequenceof IL-1ra

Forward primer2: (5′ GGCGGTCTCTAGGT-CGACCCTCTGGGAGA-3′) (SEQ ID NO:503) for cloning the mature peptide sequence of IL-1ra

In this sequence, GGTCTC is the recognition sequence of restriction enzyme Bsal.

Reverse Primer (SEQ ID NO: 504) 5′-GGCGGATCCTTA CTACTCGTCCTCCTG-3′

In this sequence, GGATCC is the recognition sequence of restriction enzyme BamHI.

1.2 Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Firstly, human IL-1Ra cDNA was obtained by reverse transcription-polymerase chain reaction techniques.

Reverse Transcription:

hepatic tissue RNA(1 μg/μl) 2 μl oligonucleotide primer 1 μl water 2 μl 5 μl Mixed, and denatured at 70° C. for 5 minutes, and then placed on ice for 5 minutes.

10X reverse transcription buffer   2 μl MgCl₂(25 mM) 4.4 μl dNTPs(10 mM)   1 μl water 6.1 μl RNase inhibitor 0.5 μl Reverse transcriptase   1 μl  15 μl

The above two solutions were mixed, and placed in the PCR machine for reverse transcription reaction (25° C. 5 minutes, 42° C. 1 hour, 70° C. 15 minutes) PCR reaction system:

10X buffer 2.5 μl cDNA Template 0.5 μl Forward primer 2 (25 μM)   2 μl Reverse primer (25 μM)   2 μl Taq DNA polymerase   1 μl dNTPs(10 mM) 0.5 μl Nuclease free water 16.5 μl  25 μl

PCR Reaction Program:

Step1 94° C. 5 min Step2 94° C. 30 sec Step3 54° C. 30 sec > 30 cycles Step4 72° C. 1 min Step5 72° C. 5 min

1.3 Purification of PCR Product

Qiagen DNA gel extraction kit was used to extract DNA from agarose gels

1. Electrophorese the PCR product through the 1% agarose gel. Locate the PCR product band which is expected to be 0.5kb in the gel using a long wavelength UV lamp.

2. Excise the gel slice containing the PCR product with clean a blade.

3. Weight the gel slice in a tube. Add 3 volume of buffer QX1 to 1 volume of gel slice.

4. Add 20 μl of QIAEX II to the sample.

5. Incubate at 50° C. for 10 min to solubilize the gel and bind DNA.

6. Centrifuge the sample for 30 sec and carefully remove the supernatant.

7. Wash the precipitate with the buffer QX1.

8. Wash the precipitate twice with the buffer PE.

9. Air-dry the precipitate for 15 min

10. Add 20 μl of H₂O and elute the DNA by vortex. Place at room temperature for 5 min

11. Centrifuge for 30 sec. Carefully transfer the supernatant into a clean tube. (The supernatant contains the purified DNA).

1.4 Restriction Enzyme Digestion:

Bsal and BamHI was used as restriction enzymes.

10× digestion buffer 2.5 μl

Bsal (10 unit/μl) 1.5 μl

Purified PCR product 21 μl

25 μl

Digested at 50oC for 1 hour. Add 0.5 μl 10× digestion buffer (NEB2), 1.5 μl BamHI (10 unit/μl), and 10× BSA (NEB) 3 μl and then digested at 37oC. for another 1 hour.

1.5 Purification of digested DNA product

Montage DNA gel extraction kit #LSKG ELO 50 (Millipore) was used.

1.6 Ligation of IL-1ra cDNA and pSUMO vector (see FIG. 1A and 1B)

1. Combine 50 μg of vector (1 μl pSUMO vector diluted with 4 μl nuclease free water to make a final concentration of 50 μg/μl) with cDNA to be inserted to a total volume of 10 μl (1 μl diluted vector plus 9 μl digested PCR product).

2. Add 10 μl of 2× ligation buffer and mix.

3. Add 1 μl of T4 DNA Ligase, mixed and centrifuged.

4. Placed at room temperature for 1 hour.

5. Move to transformation.

1.7 Transformation of E. coli DH5a

1. Thaw DH5α cells on ice.

2. 30 μl DH5α cell suspension was used per transformation.

3. 2 μl of ligated product was mixed with the 30 μl DH5α cell suspension.

4. Place the cells on ice for 45 min

5. Heat shock at 42° C. for 90 seconds in a water bath.

6. Place cells on ice for 5 minutes.

7. Add 250 μl SOC culture medium, and then shake the cells at 37° C./200 rpm for 1 hr.

8. Plate 100 μl of cells suspension on LB agar plate with 100 μg/ml ampicillin and incubate at 37° C. overnight.

1.8 Plasmid DNA Analysis

1. Pick 1-10 colonies, and PCR was conducted with a T7 forward primer and a reverse primer which is used to amplify specific IL-1ra gene to identify the positive colonies.

2. incubate 3 to 5 positive colonies into 5 ml broth medium with pg/ml ampicillin

3. Shake at 37° C./250 rpm overnight.

4. Extract plasmid DNA.

1.9 Sequencing of Plasmid DNA (see FIG. 2)

1. The extracted plasmid DNA was sequenced with a T7 forward primer and a pSUMO vector specific reverse primer.

2. After sequencing, the pSUMO expression plasmid carrying IL-1ra cDNA was stored at −80° C., and was ready to be transformed into the E. coli strain OrigamiB (DE3).

2. Expression of Interleukin-1 Receptor Antagonistic Pproteinin E. coli

Note: The following concentrations/volumes of reagents represent culture volume of 1 liter.

2.1 Transformation of E. coli OrigamiB(DE3)

1. Thaw 20 μl E. Coli origamiB(DE3) cells on ice.

2. Add 1 μl of pSUMO expression plasmid carrying IL-1ra cDNA to the competent cells, and stirred gently and mixed.

3. Tubes were placed on ice for 5 minutes.

4. Heat shock at 42° C. for 30 seconds in a water bath.

5. Place the cells on ice for 2 minutes.

6. Add 80 μl SOC medium.

7. Incubate cells at 37° C. for 1 hour.

8. Plate 100 μl cells suspension on LB/Ampicillin(100 μg/ml) agar plates and culture at 37° C. overnight.

2.2 Protein Expression

1. Medium preparation: prepare 1 liter of LB medium, preheat at 37° C. overnight, ampicillin was added before use to make a final concentration of 100 μg/ml.

2. Seed culture: pick a good colony of transformed origamiB (DE3) and incubate into a 50 ml flask containing 12.5 ml of LB medium with 100 μg/ml ampicillin, and then culture at 37° C. overnight.

3. Preparative culture: incubate 4 ml of overnight cultured origamiB (DE3) cells into 1 liter of preheated LB medium with 100 μg/ml ampicillin.

Note: strains stored in glycerol should be made and stored at −80° C. (400 μl of culture and 600 μl of sterile glycerol).

Culture samples were collected before and after IPTG induction, and analyze the protein expression by SDS-PAGE.

4. IPTG induction: after the OD600 reaches 0.4 (takes about 3 hr), induce protein expression by adding 48 mg IPTG (isopropyl-beta-D-thiogalactopyranoside) (final concentration of IPTG is 0.2 mM) and culture for additional 3 hours.

5. Cell collection: collect cells with centrifugation at 5500 rpm for 10 minutes.

2.3 Extraction: Cell Lysis

Re-suspend the cell with 15 ml of Bugbuster protein extraction reagent.

Add 15 μl Benzonase nuclease and 100 μl of 100 mg/ml PMSF (protease inhibitor) for cell lysis.

3. Incubate 15 min at room temperature with rotation.

4. Load 2 ml cell lysate into 2 ml microcentrifuge tubes.

5. Centrifuge at 14,000 rpm for 30 minutes at 4° C. in a microcentrifuge.

6. The precipitate (inclusion bodies) is solubilized in 8 ml of 50 mM HEPES-NaOH pH7.5, 6 M guanidine HCl, 25 mM DTT and left for 1 h at 4° C.

7. Insoluble material was removed by centrifugation at 14,000 rpm for 30 min at 4° C.

8. Store the supernatants (contains solubilized protein) at −20° C.

2.4 His-Tag Purification

TALON columns (Clontech, #635601, 2 ml, 2-4 mg binding capacity per column. Stored at 4° C.) was used.

Before use, flick the column to make the resin fall to the bottom.

Place column in the 2 ml microcentrifuge tube, remove the top cap and centrifuge at 1000 rpm for 2 minutes.

Add 1 ml equilibration buffer (50 mM sodium phosphate, 6M guanidineHCl, 500 mM NaCl), and fully suspend the resin.

Centrifuge the column at 1000 rpm for 2 minutes.

Repeat the equilibrating steps.

Add 1.5 ml of concentrated sample to each column.

Flick the column to suspend resin fully, and then rotate the column for 5 minutes at 4° C.

Centrifuge at 1000 rpm for 2 minutes.

Wash the column with 1 ml washing buffer (same as equilibration buffer), fully suspend the resin and rotate the column for 5 min

Centrifuge at 1000 rpm for 2 minutes.

Repeat the washing steps.

Elute the tagged protein with 700 μl elution buffer (50 mM sodium phosphate, 6M guanidineHCl, 500 mM NaCl, 150 nM imidazole) at 1000 rpm for 2 minutes.

Collect the eluate.

2.5 Protein Refolding

1. Prepare the refolding solution (50 mM HEPES pH7.5, 0.2M NaCl, 1 mM DTT, 1M NDSB201). NDSB201 is 3-(1-pyridine)-1-propene sulfonate.

2. Mix purified protein and refolding solution at a 1:5 ratio, and vortex vigorously immediately for 30 seconds.

3. Rotate the sample for 1 hr at 4° C.

4. Concentrate and dialyse the sample.

2.6 Sample Concentration

Column: Amicon Ultra-430 kDa cut-off centrifugal filter(Millipore, # UFC803024)

1. 4 ml sample was loaded on the upper portion of the Amicon centrifuge tubes.

2. Centrifuge at 3,000 rpm for 10 minutes at 4° C. or until the volume on the upper fraction reaches 1 ml.

3. Collect the concentrated sample for dialysis.

2.7 Dialysis

Equipment: D-tube Dialyzer Maxi, MWCO 6-8 (Novagen, # 71509-3)

1. Equilibrate the Dialyzer membrane by filling the dialyzer with 2 ml deionized water and place at room temperature for 5 minutes.

2. Discard the water, and load 2 ml of sample into each dialyzer.

3. Close the dialyzer and put into the floating holder.

4. Dialyze sample with 1 liter 50 mM Tris-HCl, 500 mM NaCl buffer overnight at 4° C. to remove guanidine-HCl.

5. Collect the dialyzed sample.

6. Check the protein on SDS-PAGE before His-Tag removal step.

2.8 SUMO Tag Removal

1. Add 5 μl SUMO protease into 500 μl of dialyzed IL-1ra, and incubate at 37° C. for 1 hour.

2. Remove the His-Tag using a Talon column

3. Load 100 μl of resin into an empty 1.5 ml microcentrifuge tube.

4. Centrifuge at 3,000 rpm and remove the storing buffer.

5. Add 500 μl 50 mM Tris-HCl, 500 mM NaCl to suspend and equilibrate the resin.

6. Centrifuge at 3,000 rpm, and remove the equilibrating buffer.

7. Add 500 μl digested sample into centrifuge tubes containing equilibrated resin.

8. Mix the resin and protein, rotate for 5 minutes at 4° C.

9. Centrifuge at 3000 rpm and 4° C. to separate the resin, collect the supernatant containing cleaved, separated IL-1ra.

10. Analyze the sample by 15% SDS-PAGE.

11. The sample purity was analyzed byRP-HPLC, see FIG. 4, and purify the sample with RP-HPLC, if necessary.

12. The peptide sequence could be determined by mass spectrum and peptide mass fingerprinting, see FIGS. 5 and 6.

The cDNA sequence for the insulin receptor binding polypeptide-linking group-IL-1Ra (comprising two copies of the linking group)

(SEQ ID NO: 505) TTTGTCAATCAGCACCTTTGTGGTTCTCACCTGGTGGAGGCTCTGTACCT GGTGTGTGGGGAACGTGGTTTCTTCTACACACCCAAGACCGGCTCGGGCT CGTCGTCGGCTGCTGCTCCCCAGACCGGCATTGTGGAGCAGTGCTGCACC AGCATCTGCTCCCTCTACCAACTGGAGAACTACTGCAACGGCGGCGGCGG CTCGGGCGGCGGCGGCTCGCGACCCTCTGGGAGAAAATCCAGCAAGATGC AAGCCTTCAGAATCTGGGATGTTAACCAGAAGACCTTCTATCTGAGGAAC AACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGA AAAGATAGATGTGGTACCCATTGAGCCTCATGCTCTGTTCTTGGGAATCC ATGGAGGGAAGATGTGCCTGTCCTGTGTCAAGTCTGGTGATGAGACCAGA CTCCAGCTGGAGGCAGTTAACATCACTGACCTGAGCGAGAACAGAAAGCA GGACAAGCGCTTCGCCTTCATCCGCTCAGACAGTGGCCCCACCACCAGTT TTGAGTCTGCCGCCTGCCCCGGTTGGTTCCTCTGCACAGCGATGGAAGCT GACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCAC CAAATTCACTTCCAGGAGGACGAGTAG;

The amino acid sequence of the insulin receptor binding polypeptide-linking group-IL-1Ra (comprising two copies of the linking group)(IN-7)

(SEQ ID NO: 91) FVNQHLCGSHLVEALYLVCGERGFFYTPKTGSGSSSAAAPQTGIVEQCCT SICSLYQLENYCNGGGGSGGGGSRPSGRKSSKMQAFRIWDVNQKTFYLRN NQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETR LQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEA DQPVSLTNMPDEGVMVTKFYFQEDE;

The cDNA sequence for the Exendin-4-linking group-IL-1Ra (comprising two copies of the linking group)

(SEQ ID NO: 506) CATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGA GGCAGTGCGGTTATTTATTGAGTGGCTTAAGAACGGAGGACCAAGTAGCG GGGCACCTCCGCCATCGGGCGGCGGCGGCTCGGGCGGCGGCGGCTCGCGA CCCTCTGGGAGAAAATCCAGCAAGATGCAAGCCTTCAGAATCTGGGATGT TAACCAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCTGGATACT TGCAAGGACCAAATGTCAATTTAGAAGAAAAGATAGATGTGGTACCCATT GAGCCTCATGCTCTGTTCTTGGGAATCCATGGAGGGAAGATGTGCCTGTC CTGTGTCAAGTCTGGTGATGAGACCAGACTCCAGCTGGAGGCAGTTAACA TCACTGACCTGAGCGAGAACAGAAAGCAGGACAAGCGCTTCGCCTTCATC CGCTCAGACAGTGGCCCCACCACCAGTTTTGAGTCTGCCGCCTGCCCCGG TTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCCTCACCA ATATGCCTGACGAAGGCGTCATGGTCACCAAATTCTACTTCCAGGAGGAC GAGTAG;

The amino acid sequence of the Exendin-4-linking group-IL-1Ra (comprising two copies of the linking group)(G-3)

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSRPS GRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGI HGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPG WFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:3);

The cDNA sequence for GLP-1 receptor binding polypeptide-linking group-IL-1Ra (comprising two copies of the linking group)

(SEQ ID NO: 507) CATGGTGAAGGAACATTTACCAGTGACGTGTCGTCGTACCTCGAGGAGCA GGCTGCTAAGGAGTTTATTGCTTGGCTCGTGAAGGGCCGAGGCGGCGGCG GCTCGGGCGGCGGCGGCTCGCGACCCTCTGGGAGAAAATCCAGCAAGATG CAAGCCTTCAGAATCTGGGATGTTAACCAGAAGACCTTCTATCTGAGGAA CAACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAG AAAAGATAGATGTGGTACCCATTGAGCCTCATGCTCTGTTCTTGGGAATC CATGGAGGGAAGATGTGCCTGTCCTGTGTCAAGTCTGGTGATGAGACCAG ACTCCAGCTGGAGGCAGTTAACATCACTGACCTGAGCGAGAACAGAAAGC AGGACAAGCGCTTCGCCTTCATCCGCTCAGACAGTGGCCCCACCACCAGT TTTGAGTCTGCCGCCTGCCCCGGTTGGTTCCTCTGCACAGCGATGGAAGC TGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCA CCAAATTCTACTTCCAGGAGGACGAGTAG;

The amino acid sequence of the GLP-1 receptor binding polypeptide-linking group-IL-1Ra (comprising two copies of the linking group)

(SEQ ID NO: 20) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGSGGGGSRPSGRKSSKM QAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGI HGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTS FESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE.

Construction of a Fusion Gene for GLP-1 Receptor Binding Polypeptide—Linking Group-IL-1Ra and Expression of the Fusion Protein

The cDNA fragments encoding GLP-1 and IL-1Ra were amplified by PCR procedure, using specific primers for GLP-1 and IL-1Ra (P1, GLP-1 forward primer: 5′-GGCGGTCTCTAGGTCATGGTGAAGGAACATTTA-3′ (SEQ ID NO:508); P2, GLP-1 reverse primer: 5′-CAGAGGGTCGCGAGCCGCCGCCGCCCGAGCCGCCGCCGCCTCGG CCCTTCACGA GCCA-3′ (SEQ ID NO:509); P3, IL-1Ra forward primer: 5′-GGCCGAGGCGGCGGCGGCTCGGGCGGCGGCGGCTCGCGACCCTCTGGGAGAA AA-3′ (SEQ ID NO:510); P4, IL-1Ra reverse primer: 5′-GGCGGATCCCTACTCGTCCTCCTGGAAGTAGAATTT G-3′ (SEQ ID NO:511) and the plasmids with GLP-1 and IL-1Ra genes as the templates. The restriction enzyme sites Bsa 1 and Bam HI were introduced respectively into the primers P1 and P4. The primers P2 and P3 have a complementary region of 46 base pairs in which a linking group encoding a decapeptide is contained. A fusion gene encoding GLP-1 and IL-1Ra was amplified by overlapping PCR using the primers P1 and P4 and the two above-mentioned PCR products as the templates.The fusion gene for GLP-1 and IL-1Ra were linked by two copies of the linking group GGGGS, an then the fusion gene for GLP-1 and IL-1Ra was digested with the restriction enzymes Bsa 1 and Bam HI and ligated into the vector pSUMO digested with the same enzymes, resulting in a recombinant expression plasmid comprising the fusion gene for GLP-1 and IL-1Ra. After the expression plasmid was verified by sequencing, the expression plasmid was transformed into E. coli. OrigamiB (DE3) to express the fusion protein of GLP-1 and IL-1Ra. The procedures for construction of the expression plasmid, transformation into the E. coli. OrigamiB and expression and purification of the fusion protein are all the same as those in clone and expression of the gene for interleukin-lreceptor antagonist.

The strategies for construction and expression of the fusion gene for the insulin receptor binding polypeptide-linking group-IL-1Ra, the GIP receptor binding polypeptide-linking group-IL-1Ra and Exendin-4-linking group-IL-1Ra are the same as those in construction and expression of the fusion gene for the GLP-1 receptor binding polypeptide-linking group-IL-1Ra.

The primers in the invention were synthesized by using an oligonucleotide synthesizer. The GLP-1 receptor binding polypeptide, the GIP receptor binding polypeptide, the Exendin-4 analogue, the insulin receptor binding polypeptide, and the interleukin-1 receptor antagonistic protein, even the cDNA for the full length fusion protein of the present invention, can all be synthesized chemically. Many biotechnology companies domestic and abroad provide service for synthesis of a full-length gene. The gene sequences of the cDNAs for the native human albumin and IL-1Ra can be obtained with RT-PCR from hepatic tissues of human. In addition, the cDNAs for the human albumin and IL-1Ra can be purchased from many commercial companies, such as Origene, Sino Biological Inc., etc. Methods of obtaining and expressing the cDNA for human albumin can further be referred to the literature (Lawn, et al., “The sequence of human serum albumin cDNA and its expression in E. coli.” Nucleic Acids Res. 1981, 9 (22): 6103-6114). Methods of obtaining and expressing the cDNA for IL-1Ra can be referred to the literature (Eisenberg, et al., “Primary structure and functional expression from complementary DNA of a human interleukin-1 receptor antagonist.” Nature, 1990,343: 341-346).

The cDNA seauence for Human albumin

(SEQ ID NO: 512) GATGCAC ACAAGAGTGA GGTTGCTCAT CGGTTTAAAG ATTTGGGAGA AGAAAATTTC AAAGCCTTGGTGTTGATTGC CTTTGCTCAG TATCTTCAGC AGTGTCCATT TGAAGATCAT GTAAAATTAGTGAATGAAGT AACTGAATTT GCAAAAACAT GTGTTGCTGA TGAGTCAGCT GAAAATTGTGACAAATCACT TCATACCCTT TTTGGAGACA AATTATGCAC AGTTGCAACT CTTCGTGAAACCTATGGTGA AATGGCTGAC TGCTGTGCAA AACAAGAACC TGAGAGAAAT GAATGCTTCTTGCAACACAA AGATGACAAC CCAAACCTCC CCCGATTGGT GAGACCAGAG GTTGATGTGATGTGCACTGC TTTTCATGAC AATGAAGAGA CATTTTTGAA AAAATACTTA TATGAAATTGCCAGAAGACA TCCTTACTTT TATGCCCCGG AACTCCTTTT CTTTGCTAAA AGGTATAAAGCTGCTTTTAC AGAATGTTGC CAAGCTGCTG ATAAAGCTGC CTGCCTGTTG CCAAAGCTCGATGAACTTCG GGATGAAGGG AAGGCTTCGT CTGCCAAACA GAGACTCAAG TGTGCCAGTCTCCAAAAATT TGGAGAAAGA GCTTTCAAAG CATGGGCAGT AGCTCGCCTG AGCCAGAGATTTCCCAAAGC TGAGTTTGCA GAAGTTTCCA AGTTAGTGAC AGATCTTACC AAAGTCCACACGGAATGCTG CCATGGAGAT CTGCTTGAAT GTGCTGATGA CAGGGCGGAC CTTGCCAAGTATATCTGTGA AAATCAAGAT TCGATCTCCA GTAAACTGAA GGAATGCTGT AAAAACCTCTGTTGGAAAA ATCCCACTGC ATTGCCGAAG TGGAAAATGA TGAGATGCCT GCTGACTTGCCTTCATTAGC TGCTGATTTT GTTGAAAGTA AGGATGTTTG CAAAAACTAT GCTGAGGCAAAGGATGTCTT CCTGGGCATG TTTTTGTATG AATATGCAAG AAGGCATCCT GATTACTCTGTCGTGCTGCT GCTGAGACTT GCCAAGACAT ATGAAACCAC TCTAGAGAA TGCTGTGCCGCTGCAGATCC TCATGAATGC TATGCCAAAG TGTTCGATGA ATTTAAACCT CTTGTGGAAGAGCCTCAGAA TTTAATCAAA CAAAATTGTG AGCTTTTTGA GCAGCTTGGA GAGTACAAATTCCAGAATGC GCTATTAGTT CGTTACACCA AGAAAGTACC CCAAGTGTCA ACTCCAACTCTTGTAGAGG CTCAAGAAAC CTAGGAAAAG TGGGCAGCAA ATGTTGTAAA CATCCTGAAGCAAAAAGAAT GCCCTGTGCA GAAGACTATC TATCCGTGGT CCTGAACCAG TTATGTGTGTTGCATGAGAA AACGCCAGTA AGTGACAGAG TCACCAAATG CTGCACAGAA TCCTTGGTGAACAGGCGACC ATGCTTTTCA GCTCTGGAAG TCGATGAAAC ATACGTTCCC AAAGAGTTTAATGCTGAAAC GTTCACCTTC CATGCAGATA TATGCACACT TTCTGAGAAG GAGAGACAAATCAAGAAACA AACTGCACTT GTTGAGCTTG TGAAACACAA GCCCAAGGCA ACAAAAGAGCAACTGAAAGC TGTTATGGAT GATTTCGCAG CTTTTGTAGA GAAGTGCTGC AAGGCTGACGATAAGGAGAC CTGCTTTGCC GAGGAGGGTA AAAAACTTGT TGCTGCAAGT CAAGCTGCCTTAGGCTTATA A.

The amino acid sequence of human albumin

(SEQ ID NO: 513) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFA KTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNE CFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFY APELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKC ASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDL LECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPA DLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLA KTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAE DYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPK EFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDD FAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL.

The strategies for connection of human albumin to several above-mentioned fusion genes, the construction, and expression of the fusion protein are the same as those in construction of the fusion gene for the GLP-1 receptor binding polypeptide-linking group-IL-1Ra and expression of the fusion protein.

The cDNA sequence for the GIP-linking group-IL-1Ra (comprising two copies of the linking group)

(SEQ ID NO: 514) TACGCGGAAGGGACTTTCATCAGTGACTACAGTATTGCCATGGACAAGAT TCACCAACAAGACTTTGTGAACTGGCTGCTGGCCCAAAAGGGGAAGAAGA ATGACTGGAAACACAACATCACCCAGGGCGGCGGCGGCTCGGGCGGCGGC GGCTCGCGACCCTCTGGGAGAAAATCCAGCAAGATGCAAGCCTTCAGAAT CTGGGATGTTAACCAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTG CTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGATAGATGTG GTACCCATTGAGCCTCATGCTCTGTTCTTGGGAATCCATGGAGGGAAGAT GTGCCTGTCCTGTGTCAAGTCTGGTGATGAGACCAGACTCCAGCTGGAGG CAGTTAACATCACTGACCTGAGCGAGAACAGAAAGCAGGACAAGCGCTTC GCCTTCATCCGCTCAGACAGTGGCCCCACCACCAGTTTTGAGTCTGCCGC CTGCCCCGGTTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCA GCCTCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTCTACTTC CAGGAGGACGAGTAG.

The amino acid sequence of the GIP receptor binding polypeptide-linking group-IL-1Ra(comprising two copies of the linking group)(non-acylated GI-4 at N-terminal)

(SEQ ID NO: 158) YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGGG GSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDV VPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRF AFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYF QEDE. (2,5-dioxopyrrolidin-1-yl)acetate

Acetic acid (1.5 g, 25 mM) was dissolved in methylene chloride (15 ml), DCC (5.16 g, 25 mM) and a solution of N-hydroxyl butanimide (2.88 g, 25 mM) in dioxan (5 ml) was added, stirred at 4° C. overnight, and at room temperature for 2 h. The mixture was filtered, the solvent was evaporated under a reduced pressure, petroleum ether (b.p. 60-80° C.) was added, the crystal was filtered and recrystallized in ethyl acetate to yield a colourless needle product (3.75 g, yield: 95.5%), m.p. 131-134° C.

Acylation of the N-Terminus of the GIP Fusion Protein

The single chain fusion protein of GIP receptor binding polypeptide and interleukin-1 receptor antagonistic protein was prepared according to the above procedure. With respect to the fusion proteins which need to be acylated at the N-terminus, such as GI-2 to GI-5, GI-13 to 15, GI-22, etc., the fusion proteins were dissolved in 0.01 NHCl, the resultant solution was adjusted with NaOH to pH 6.9, the equimolar amount of (2,5-dioxopyrrolidin-1-yl) acetate salt was added with one tenth of the amount added each time and the alkali solution was added every 30 mM to keep at pH 6.9. The reaction was stirred overnight and purified on RP-HPLC C18 column. Buffer A: 0.1% TFA in aqueous solution, 10% acetonitrile; buffer B: 0.1% TFA in aqueous solution, 80% acetonitrile.

With the above-mentioned method, single chain fusion proteins of the GLP-1 receptor binding polypeptide and the interleukin-1 receptor antagonistic protein were prepared, their molecular weights were determined with mass spectrometry, and the synthesized compounds were verified by determining the structure of the single chain polypeptide through sequencing, with the results as follows:

G-1: the calculated molecular weight value thereof is 21296.0 and the molecular weight determined by mass spectrometry is 21298.3, with the sequencing result consistent with the sequence shown in the instant application;

G-2: the calculated molecular weight value thereof is 21611.3 and the molecular weight determined by mass spectrometry is 21613.4, with the sequencing result consistent with the sequence shown in the instant application;

G-3: the calculated molecular weight value thereof is 21926.5 and the molecular weight determined by mass spectrometry is 21928.1, with the sequencing result consistent with the sequence shown in the instant application;

G-4: the calculated molecular weight value thereof is 22241.8 and the molecular weight determined by mass spectrometry is 22243.2, with the sequencing result consistent with the sequence shown in the instant application;

G-5: the calculated molecular weight value thereof is 20518.1 and the molecular weight determined by mass spectrometry is 20520.6, with the sequencing result consistent with the sequence shown in the instant application;

G-6: the calculated molecular weight value thereof is 20719.3 and the molecular weight determined by mass spectrometry is 20721.0, with the sequencing result consistent with the sequence shown in the instant application;

G-7: the calculated molecular weight value thereof is 21034.6 and the molecular weight determined by mass spectrometry is 21035.8, with the sequencing result consistent with the sequence shown in the instant application;

G-8: the calculated molecular weight value thereof is 21349.9 and the molecular weight determined by mass spectrometry is 21351.7, with the sequencing result consistent with the sequence shown in the instant application;

G-18: the calculated molecular weight value thereof is 20522.1 and the molecular weight determined by mass spectrometry is 20524.3, with the sequencing result consistent with the sequence shown in the instant application;

G-19: the calculated molecular weight value thereof is 20780.3 and the molecular weight determined by mass spectrometry is 20781.8, with the sequencing result consistent with the sequence shown in the instant application;

G-20: the calculated molecular weight value thereof is 21095.6 and the molecular weight determined by mass spectrometry is 21098.0, with the sequencing result consistent with the sequence shown in the instant application;

G-21: the calculated molecular weight value thereof is 21410.9 and the molecular weight determined by mass spectrometry is 21412.4, with the sequencing result consistent with the sequence shown in the instant application;

G-22: the calculated molecular weight value thereof is 21873.5 and the molecular weight determined by mass spectrometry is 21875.6, with the sequencing result consistent with the sequence shown in the instant application;

G-81: the calculated molecular weight value thereof is 89011.3 and the molecular weight determined by mass spectrometry is 89013.6, with the sequencing result consistent with the sequence shown in the instant application;

G-82: the calculated molecular weight value thereof is 88958.2 and the molecular weight determined by mass spectrometry is 88960.1, with the sequencing result consistent with the sequence shown in the instant application;

G-83: the calculated molecular weight value thereof is 89011.3 and the molecular weight determined by mass spectrometry is 89013.0, with the sequencing result consistent with the sequence shown in the instant application;

G-84: the calculated molecular weight value thereof is 88958.2 and the molecular weight determined by mass spectrometry is 88960.5, with the sequencing result consistent with the sequence shown in the instant application.

With the above-mentioned method, fusion proteins were prepared on the basis of insulin receptor binding polypeptide and interleukin-1 receptor antagonistic protein, their molecular weights were determined with mass spectrometry, and the synthesized compounds were verified by determining the structure of the single chain polypeptide through sequencing, with the results as follows:

IN-1: the calculated molecular weight value thereof is 23900.0 and the molecular weight determined by mass spectrometry is 23902.8, with the sequencing result consistent with the sequence shown in the instant application;

IN-2: the calculated molecular weight value thereof is 23884.0 and the molecular weight determined by mass spectrometry is 23886.7, with the sequencing result consistent with the sequence shown in the instant application;

IN-3: the calculated molecular weight value thereof is 23835.8 and the molecular weight determined by mass spectrometry is 23837.2, with the sequencing result consistent with the sequence shown in the instant application;

IN-4: the calculated molecular weight value thereof is 24215.3 and the molecular weight determined by mass spectrometry is 24317.5, with the sequencing result consistent with the sequence shown in the instant application;

IN-5: the calculated molecular weight value thereof is 24199.2 and the molecular weight determined by mass spectrometry is 24151.3, with the sequencing result consistent with the sequence shown in the instant application;

IN-6: the calculated molecular weight value thereof is 24151.1 and the molecular weight determined by mass spectrometry is 24153.7, with the sequencing result consistent with the sequence shown in the instant application;

IN-7: the calculated molecular weight value thereof is 24530.6 and the molecular weight determined by mass spectrometry is 24532.4, with the sequencing result consistent with the sequence shown in the instant application;

IN-8: the calculated molecular weight value thereof is 24514.5 and the molecular weight determined by mass spectrometry is 24517.1, with the sequencing result consistent with the sequence shown in the instant application;

IN-9: the calculated molecular weight value thereof is 24466.4 and the molecular weight determined by mass spectrometry is 24469.0, with the sequencing result consistent with the sequence shown in the instant application;

IN-10: the calculated molecular weight value thereof is 24845.9 and the molecular weight determined by mass spectrometry is 24847.3, with the sequencing result consistent with the sequence shown in the instant application;

IN-11: the calculated molecular weight value thereof is 24829.8 and the molecular weight determined by mass spectrometry is 24830.9, with the sequencing result consistent with the sequence shown in the instant application;

IN-12: the calculated molecular weight value thereof is 24781.6 and the molecular weight determined by mass spectrometry is 24783.0, with the sequencing result consistent with the sequence shown in the instant application;

IN-13: the calculated molecular weight value thereof is 23309.3 and the molecular weight determined by mass spectrometry is 23311.2, with the sequencing result consistent with the sequence shown in the instant application;

IN-14: the calculated molecular weight value thereof is 23293.3 and the molecular weight determined by mass spectrometry is 23295.1, with the sequencing result consistent with the sequence shown in the instant application;

IN-15: the calculated molecular weight value thereof is 23245.1 and the molecular weight determined by mass spectrometry is 23246.8, with the sequencing result consistent with the sequence shown in the instant application;

IN-16: the calculated molecular weight value thereof is 23624.6 and the molecular weight determined by mass spectrometry is 23626.5, with the sequencing result consistent with the sequence shown in the instant application;

IN-17: the calculated molecular weight value thereof is 23608.6 and the molecular weight determined by mass spectrometry is 23610.0, with the sequencing result consistent with the sequence shown in the instant application;

IN-18: the calculated molecular weight value thereof is 23560.4 and the molecular weight determined by mass spectrometry is 23561.7, with the sequencing result consistent with the sequence shown in the instant application;

IN-19: the calculated molecular weight value thereof is 23939.9 and the molecular weight determined by mass spectrometry is 23941.6, with the sequencing result consistent with the sequence shown in the instant application;

IN-20: the calculated molecular weight value thereof is 23923.9 and the molecular weight determined by mass spectrometry is 23925.3, with the sequencing result consistent with the sequence shown in the instant application;

IN-21: the calculated molecular weight value thereof is 23875.7 and the molecular weight determined by mass spectrometry is 23877.2, with the sequencing result consistent with the sequence shown in the instant application;

IN-22: the calculated molecular weight value thereof is 24255.2 and the molecular weight determined by mass spectrometry is 24256.9, with the sequencing result consistent with the sequence shown in the instant application;

IN-23: the calculated molecular weight value thereof is 24239.1 and the molecular weight determined by mass spectrometry is 24241.5, with the sequencing result consistent with the sequence shown in the instant application;

IN-24: the calculated molecular weight value thereof is 24191.0 and the molecular weight determined by mass spectrometry is 24192.6, with the sequencing result consistent with the sequence shown in the instant application;

IN-62: the calculated molecular weight value thereof is 24530.6 and the molecular weight determined by mass spectrometry is 24537.1, with the sequencing result consistent with the sequence shown in the instant application;

IN-63: the calculated molecular weight value thereof is 24514.5 and the molecular weight determined by mass spectrometry is 24525.5, with the sequencing result consistent with the sequence shown in the instant application;

IN-64: the calculated molecular weight value thereof is 24466.4 and the molecular weight determined by mass spectrometry is 24474.7, with the sequencing result consistent with the sequence shown in the instant application;

IN-65: the calculated molecular weight value thereof is 24503.6 and the molecular weight determined by mass spectrometry is 24515.4, with the sequencing result consistent with the sequence shown in the instant application;

IN-66: the calculated molecular weight value thereof is 25064.2 and the molecular weight determined by mass spectrometry is 25073.6, with the sequencing result consistent with the sequence shown in the instant application;

IN-67: the calculated molecular weight value thereof is 24933.0 and the molecular weight determined by mass spectrometry is 24942.0, with the sequencing result consistent with the sequence shown in the instant application;

IN-68: the calculated molecular weight value thereof is 91615.4 and the molecular weight determined by mass spectrometry is 91624.9, with the sequencing result consistent with the sequence shown in the instant application;

IN-69: the calculated molecular weight value thereof is 91615.4 and the molecular weight determined by mass spectrometry is 91627.2, with the sequencing result consistent with the sequence shown in the instant application;

IN-70: the calculated molecular weight value thereof is 91730.6 and the molecular weight determined by mass spectrometry is 91623.3, with the sequencing result consistent with the sequence shown in the instant application.

With the above-mentioned method, fusion proteins were prepared on the basis of the GIP receptor binding polypeptide and the interleukin-1 receptor antagonistic protein, their molecular weights were determined with mass spectrometry, and the synthesized compounds were verified by determining the structure of the single chain polypeptide through sequencing, with the results as follows:

GI-1: the calculated molecular weight value thereof is 22423.2 and the molecular weight determined by mass spectrometry is 22427.0, with the sequencing result consistent with the sequence shown in the instant application;

GI-2: the calculated molecular weight value thereof is 22134.0 and the molecular weight determined by mass spectrometry is 22138.1, with the sequencing result consistent with the sequence shown in the instant application;

GI-3: the calculated molecular weight value thereof is 22449.2 and the molecular weight determined by mass spectrometry is 22456.3, with the sequencing result consistent with the sequence shown in the instant application;

GI-4: the calculated molecular weight value thereof is 22764.5 and the molecular weight determined by mass spectrometry is 22772.4, with the sequencing result consistent with the sequence shown in the instant application;

GI-5: the calculated molecular weight value thereof is 23079.8 and the molecular weight determined by mass spectrometry is 23090.7, with the sequencing result consistent with the sequence shown in the instant application;

GI-6: the calculated molecular weight value thereof is 22060.0 and the molecular weight determined by mass spectrometry is 22068.5, with the sequencing result consistent with the sequence shown in the instant application;

GI-7: the calculated molecular weight value thereof is 22375.2 and the molecular weight determined by mass spectrometry is 22384.6, with the sequencing result consistent with the sequence shown in the instant application;

GI-8: the calculated molecular weight value thereof is 22690.5 and the molecular weight determined by mass spectrometry is 22695.9, with the sequencing result consistent with the sequence shown in the instant application;

GI-9: the calculated molecular weight value thereof is 23005.8 and the molecular weight determined by mass spectrometry is 23009.2, with the sequencing result consistent with the sequence shown in the instant application;

GI-10: the calculated molecular weight value thereof is 21381.0 and the molecular weight determined by mass spectrometry is 21373.4, with the sequencing result consistent with the sequence shown in the instant application;

GI-11: the calculated molecular weight value thereof is 22351.0 and the molecular weight determined by mass spectrometry is 22360.8, with the sequencing result consistent with the sequence shown in the instant application;

GI-12: the calculated molecular weight value thereof is 21720.4 and the molecular weight determined by mass spectrometry is 21724.1, with the sequencing result consistent with the sequence shown in the instant application;

GI-13: the calculated molecular weight value thereof is 22407.0 and the molecular weight determined by mass spectrometry is 22411.5, with the sequencing result consistent with the sequence shown in the instant application;

GI-14: the calculated molecular weight value thereof is 22091.7 and the molecular weight determined by mass spectrometry is 22097.6, with the sequencing result consistent with the sequence shown in the instant application;

GI-15: the calculated molecular weight value thereof is 21776.5 and the molecular weight determined by mass spectrometry is 21782.4, with the sequencing result consistent with the sequence shown in the instant application;

GI-16: the calculated molecular weight value thereof is 20750.4 and the molecular weight determined by mass spectrometry is 20753.9, with the sequencing result consistent with the sequence shown in the instant application;

GI-17: the calculated molecular weight value thereof is 20435.1 and the molecular weight determined by mass spectrometry is 20438.7, with the sequencing result consistent with the sequence shown in the instant application;

GI-18: the calculated molecular weight value thereof is 21528.2 and the molecular weight determined by mass spectrometry is 21534.2, with the sequencing result consistent with the sequence shown in the instant application;

GI-19: the calculated molecular weight value thereof is 20609.3 and the molecular weight determined by mass spectrometry is 20613.3, with the sequencing result consistent with the sequence shown in the instant application;

GI-20: the calculated molecular weight value thereof is 21702.5 and the molecular weight determined by mass spectrometry is 21709.0, with the sequencing result consistent with the sequence shown in the instant application;

GI-21: the calculated molecular weight value thereof is 22017.7 and the molecular weight determined by mass spectrometry is 22025.6, with the sequencing result consistent with the sequence shown in the instant application;

GI-22: the calculated molecular weight value thereof is 89218.7 and the molecular weight determined by mass spectrometry is 89227.1, with the sequencing result consistent with the sequence shown in the instant application;

GI-23: the calculated molecular weight value thereof is 89144.7 and the molecular weight determined by mass spectrometry is 89153.5, with the sequencing result consistent with the sequence shown in the instant application.

Chemical Synthesis of Polypeptides

Linear polypeptides can be synthesized by Boc solid phase polypeptide synthesis or Fmoc solid phase polypeptide synthesis. If a polypeptide with a carboxyl group at the C-terminus is chemically synthesized by Fmoc method, Wang resin would be selected, while if a polypeptide with an amide at the C-terminus, Rink amide resin would be selected. If a polypeptide with a carboxyl group at the C-terminus is chemically synthesized by Boc method, Pam resin would be selected, while if a polypeptide with an amide at the C-terminus, MBHA resin would be selected. The condensation agents and activating agents are DIC and HOBT. Other alternative condensation agents include BOP, HBTU, DEPBT, etc Amino acids are used 5 folds in excess and the time period for condensation is 1 hour.

Fmoc protective group can be removed by 50% piperidine/DMF and Boc protective group can be cleaved by TFA. Peptide bond condensation can be monitored by ninhydrin (2,2-Dihydroxyindane-1,3-dione) test.

In Fmoc solid phase polypeptide synthesis, the commonly used amino acids and protective groups are as follows:

Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Boc-Trp(Boc)-OH or Fmoc-Tyr(tBu)-OH.

If the side chain amino group of lysine is used for acylation, side chain amino group of lysine could be protected by allyloxycarbonyl (aloc) group. Upon completion of synthesis of peptide sequence, allyloxycarbonyl (aloc) group can be removed using tetrakis(triphenylphosphine)palladium(0) along with a 37:2:1 mixture of DCM, acetic acid, and NMM (15 ml/g resin) in an argon atmosphere by stirring at ambient temperature for 2 hours. After reaction, the resin should be washed with 0.5% DIPEA/DMF(10 ml), 0.5% sodium diethyldithiocarbamate trihydrate/DMF (3×10 ml), and 1:1 DCM:DMF (5×10 ml). The side chain amino group of lysine may also be protected by 4-methyltrityl group (Mtt). The resin is suspended in DCM, followed by addition of TFA/TIS/DCM(1:2:97),vibrated for 10 minutes, repeated twice, and then washed with DCM, DMF and isopropyl alcohol.

After a polypeptide is synthesized by Fmoc solid phase synthesis, the cleavage reagent commonly used is a TFA. The dry resin was placed into a shaking flask, filled with an appropriate amount of TFA/TIS/H₂O(95:2.5: 2.5, 10-25 mL/g resin), capped with a cover, and subjected to batch-type rotary shaking at room temperature. After 2 h, the resin was subjected to suction filtration and washed 2-3 times with fresh TFA; the filtrates were pooled, and 8-10 volumes of iced ethyl ether was added dropwise. Finally, the precipitated polypeptide crude was collected by centrifugation.

In Boc solid phase polypeptide synthesis, the commonly used amino acids and protective groups are as follows: Boc-Cys(4-MeBzl)-OH, Boc-Asp(OcHx)-OH, Boc-Glu(OcHx)-OH, Boc-His(Bom)-OH, Boc-Lys(2-Cl—Z)—OH, Boc-Asn(Xan)-OH, Boc-Arg(Tos)-OH, Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Trp(CHO)—OH and Boc-Tyr(2-Br—Z)—OH.

If the side chain amino group of a lysine is used for formation of lactam or acylation reaction, side chain amino group of lysine may be protected by allyloxycarbonyl (aloc) or Fmoc group. If carboxyl of the side chain of glutamic acid or aspartic acid is used for formation of lactam or acylation reaction, the carboxyl may be converted to an allyl ester or be protected by 9-Fluorenylmethyl group.

After a polypeptide is synthesized by solid phase Boc synthesis, PAM and MBHA resins are usually cleaved by HF. For 0.1 mM resin, 5 ml of HF, together with p-cresol, p-hydroxy thiophenol, or anisole etc are added. The mixture is stirred at ice bath temperature for 1 hour. After HF is evaporated in vacuo, the polypeptide is precipitated with iced ethyl ether and collected by centrifugation. The final product is obtained after HPLC purification and subjected to freeze drying.

Preparation of Tert-Butyl Hexadecandioyl-L-Glu(OSu)-OtBu

5.72 g (20 mM) of hexadecanedioic acid was dissolved in 240 mL of anhydrous DMF and cooled in an ice bath. 1.48 g (20 mM) of 2-methyl-2-propyl alcohol, 2.7 g (2.25 mL, 21.4 mM) of DIC, 2.88 g (21.4 mM) of HOBT, 2.16 g (2.34 mL, 21.4 mM) of NMM, and 244 mg (2 mM) of DMAP were addedsuccessively. The mixture was stirred at room temperature overnight. 80 mL of water was added, acidified to pH 3, and extracted with ethyl acetate (80 mL×3). The organic layer was washed with 0.1 NHCl and saturated saline, dried over magnesium sulphate, and the solvent was evaporated under a reduced pressure to obtain mono-tert-butyl hexadecandioate (3.32 g, yield: 47%). Data for nuclear magnetic resonance were ¹H-NMR (CDCl₃) δ 2.35 (t, 2H), 1.56-1.66 (m, 4H), 1.44 (s, 9H), 1.21-1.35 (m, 20H).

4.25 g (10 mM) of Fmoc-Glu-OtBu was dissolved in 30 mL of DCM, transferred into 3 g of 2-CTC resin (2-chlorotritylchloride resin, sub. 1 mM/g), and followed by adding 1.29 g (10 mM, 1.74 mL) of DIPEA. After the mixture was shaken in a shaker for 5 mM, 1.93 g (15 mM, 2.6 mL) of DIPEA was further added. The mixture was shaken vigorously for one hour. To the resin was added 2.4 mL of HPLC-grade methanol and mixed for 15 mM The resin was filtered, washed with DCM (3×30 mL), DMF (2×30 mL), DCM (3×30 mL), methanol (3×30 mL) and then desiccated in vacuum.

After Fmoc was deprotected with piperidine, 3 g of the resin (3 mM) and 3.43 g (10 mM) of mono-tert-butyl hexadecandioate were added to 50 mL of anhydrous DMF, and 1.35 g (1.12 mL, 10.7 mM) of DIC, 1.44 g (10.7 mM) of HOBT, 1.3 g (10 mM, 1.74 mL) of DIPEA were added successively. After agitated at room temperature overnight, the resin was rinsed with DMF (2×30 mL) and DCM (2×30 mL).

A cleavage solution of AcOH/TFE/DCM (1:1:8) (20 mL/g resin) was prepared. The resin was suspended in half of the cleavage solution, and placed at room temperature for 30 mM The resin was filtered and washed three times with another half of the cleavage solution. The filtrates were mixed and 15 volumes of n-hexane were added. Excessive acetic acid was removed by rotary evaporation to yield tert-butyl hexadecandioyl -L-Glu-OtBu. ¹H-NMR(CDCl₃): δ 6.25(d, 1H), 4.53 (m, 1H), 2.42 (m, 2H), 2.21 (m, 4H), 1.92 (m, 1H), 1.58 (m, 4H), 1.47 (s, 9H), 1.22-1.43 (m, 18H).

1 g (1.9 mM) of tert-butyl hexadecandioyl-L-Glu-OtBu was dissolved in 5 mL of anhydrous DMF/DCM (1 mL:4 mL). 0.412 g (2 mM) of DCC and 0.23 g (2 mM) of N-hydroxyl succinimide were added. The mixture was stirred at room temperature overnight. The mixture was filtered. The filtrate was diluted with ethyl acetate, washed with 0.1 NHCl and saturated saline, dried over magnesium sulphate, and evaporated under a reduced pressure to obtain tert-butyl hexadecandioyl-L-Glu (OSu)-OtBu. ¹H-NMR(CDCl₃): δ 6.17 (d, 1H), 4.60 (m, 1H), 2.84 (s, 4H), 2.72 (m, 1H), 2.64 (m, 1H), 2.32 (m, 1H), 2.20 (m, 4H), 2.08 (m, 1H), 1.6 (m, 4H), 1.47 (s, 9H), 1.43 (s, 9H), 1.20-1.33 (m, 20H).

Tert-butyl octadecandioyl-L-Glu(OSu)-OtBu was prepared in the same procedure.

Acylation of the protein/polypeptide (1)

The single chain insulin analogue (10 μmol) was dissolved in 0.01 NHCl at room temperature, and 0.01 NNaOH was added dropwise to pH 7. Tert-butyl hexadecandioyl-L-Glu (OSu)-OtBu (12 μmol) was dissolved in acetonitrile (2 mL), and the solution of polypeptide was added. After stirred for 30 mM, the mixture was acidified with 50% acetic acid, and loaded onto a RP-HPLC C5 column for purification. Buffer A: 0.1% TFA aqueous solution, 10% acetonitrile; buffer B: 0.1% TFA aqueous solution, 80% acetonitrile. The polypeptide initially purified and lyophilized was placed into TFA/TIS/H₂O (95:2.5:2.5, 10 mL). Thirty minutes later, the solvent was evaporated under a reduced pressure and the crude product was dissolved in Buffer A and lyophilized Purification was performed using the RP-HPLC C5 column, with buffer A: 0.1% TFA aqueous solution, 10% acetonitrile and buffer B: 0.1% TFA aqueous solution, 80% acetonitrile. The acylated single chain insulin analogue was dissolved in 100 mM Na₂CO₃ (2 mL). Maleimide-PEG12-NHS (12 μmol) was added and stirred for 30 mM, interleukin-1 receptor antagonistic protein (11 μmol) was added. After stirred for 5 hours, the mixture was acidified with 50% acetic acid, and loaded onto a RP-HPLC C5 column for purification. Buffer A: 0.1% TFA aqueous solution, 10% acetonitrile; buffer B: 0.1% TFA aqueous solution, 80% acetonitrile.

With the above-mentioned method, fusion proteins were prepared on the basis of the insulin receptor binding polypeptide and the interleukin-1 receptor antagonistic protein, and their molecular weights were determined with mass spectrometry as follows:

IN-33: the calculated molecular weight value thereof is 25135.4 and the molecular weight determined by mass spectrometry is 25136.9, with the sequencing result consistent with the sequence shown in the instant application;

IN-34: the calculated molecular weight value thereof is 25108.4 and the molecular weight determined by mass spectrometry is 25110.5, with the sequencing result consistent with the sequence shown in the instant application.

Synthesis of 18-Maleimidooctadecanoic Acid

Methyl 18-hydroxyoctadecanoate (4.5 g, 14.3 mM) was dissolved in50 ml dichloromethane. Pyridine (4.53 g, 57.2 mM) was added, and cooled in a ice bath, and then p-toluenesulfonyl chloride (5.46 g, 28.6 mM) was slowly added within 1 hour. Thereafter, reaction was carried out for 16 hours under stir and 4° C. The reaction mixture was washed with a 1N hydrochloric acid aqueous solution, water, saturated sodium hydrogen carbonate solution, water and saturated saline solution successively, dried, and then concentrated under a reduced pressure. Then purified by silica gel column (eluent: benzene) to give methyl 18-tosyloxyoctadecanoate (5.69 g, 85%). m.p. 67.5-68.5° C. ¹H-NMR (CDCl₃, 270 MHz): δ 1.09-1.42 (m, 26H), 1.53-1.72 (m, 4H), 2.30 (t, 2H), 2.45 (s, 3H), 3.68 (s, 3H), 4.03 (t, 2H), 7.35 (d, 2H), 7.79 (d, 2H).

A mixture of potassium phthalimide (2.96 g, 16.0 mM) and 100 ml anhydrous DMF was heated to 110° C. 80 ml methyl 18-tosyloxyoctadecanoate (5.0 g, 10.7 mM) dissolved in DMF was added dropwise and the mixture was stirred at 110° C. for 2 hours. Ice water was poured to reach a total volume of 1.2 liter, and stirred for 30 minutes. Thereafter, the precipitates were filtered and dissolved in chloroform, and then washed with water and saturated saline solution, dried over magnesium sulfate, concentrated under a reduced pressure and purified by silica gel column (eluent: benzene and methylene chloride: 2:1), to give methyl 18-phtahlimideocatadecanoate (4.15 g, 88%).m.p. 82-83° C.

¹H-NMR (CDCl₃, 270 MHz): δ 1.09-1.42 (m, 26H), 1.53-1.76 (m, 4H), 2.30 (t,2H), 3.66, 3.67 (s, t, 5H), 7.71 (m, 2H), 7.84 (m, 2H).

Methyl 18-phthalimideoctadecanoate (2.0 g, 4.51 mM), 30 ml ethanol and 80% hydrazine hydrate (0.42 ml, 6.76 mM) were heated and refluxed for 9 hours. 6N hydrochloric acid (11.3 ml, 67.6 mM) was added, heated and refluxed for 1 hour. The insoluble matter was filtered off, and the filtrate was concentrated under a reduced pressure. 30 ml of ethanol and 18.1 ml of 1N sodium hydroxide solution was added, heated and refluxed for 18 hours. The reaction was neutralized with 6N hydrochloric acid. The precipitates were collected by filtration and recrystallized in ethanol-acetic acid-water to give 18-aminooctadecanoic acid (800 mg, 59%). m.p. 172°-174° C. IR (cm⁻¹): 2920, 2850, 1640, 1535, 1470, 1400 FD-MS (m/z): [M+H]⁺ 300.

18-aminooctadecanoic acid (400 mg, 1.33 mM)was dissolved in 50 ml of ethanol and 25 ml of 1N sodium hydroxide, at 40° C. At the same temperature, maleic anhydride (1.97 g, 20.0 mM) was slowly added within 2 hours. The reaction mixture was stirred for 30 minutes, then acidified with hydrochloric acid and subjected to centrifugation. The precipitate was filtered and washed with water, and then dried under a reduced pressure, to give N-(17-carboxyheptadecyl)maleamic acid (436 mg, 88%). m.p. 144°-147.5° C. IR (cm⁻¹): 3305, 2920, 2850, 1710, 1630, 1585, 1470, 1400, 1280, 1250, 1230, 1215, 1195, 1180.

FD-MS (m/z): [M+H]⁺ 398

N-(17-carboxyheptadecyl)maleamic acid (400 mg, 1.01 mM), 2.83 mlacetic anhydride, and anhydrous sodium acetate (41.0 rag, 0.50 mM) was heated to 100° C., stirred for 1 hour. After cooling of the reaction, ice was added and further stirred for 1 hour. The resulting mixture was extracted with chloroform. The organic layer was washed with water and then with saturated saline solution, dried over anhydrous magnesium sulfate, concentrated under a reduced pressure and purified by silica gel column (eluent: benzene and chloroform: 1:1), to give 18-maleimidooctadecanoic acid (172 mg, 45%). m.p. 101-103° C.

¹H-NMR (CDCl₃, 270 MHz): δ 1.14-1.40 (m, 26H), 1.48-1.72 (m, 4H), 2.35 (t, 2H), 3.50 (t, 2H), 6.68 (s, 2H)

IR(cm⁻¹): 2920, 2850, 1710, 1470, 1450, 1410, 840, 700 FD-MS (m/z): [M+H]⁺ 380

Acylation of the protein/ polypeptide (2): modification of the fusion protein by 18-maleimidooctadecanoic acid 3 ml aqueous solution of fusion protein (3 mg) was mixed with 0.8 ml of 0.5 M Tris-HCl buffer (pH 9), 18-maleimidooctadecanoic acid was added slowly (in a molar ratio of 1.1). The reaction was stirred at room temperature overnight. The reaction was filtered, the filtrate was added onto Sephadex G-25 (the eluent: 10 mM ammonium bicarbonate solution). Fractions with large molecular weights were collected, and purified with a DEAE-Sepharose Fast Flow ion exchange column (the eluent: 10 mM Tris-HCl (pH 8) and 0.075 M sodium chloride solution). The collected product was desalted with Sephadex G-25 (the eluent: 10 mM ammonium bicarbonate solution) and freeze dried to obtain the product.

With the above-mentioned method, fusion proteins were prepared on the basis of the GLP-1 receptor binding polypeptide and the interleukin-1 receptor antagonistic protein, and their molecular weights were determined with mass spectrometry as follows:

G-30: the calculated molecular weight value thereof is 22278.0 and the molecular weight determined by mass spectrometry is 22279.5, which is consistent with the compound shown in the instant application;

G-31: the calculated molecular weight value thereof is 22306.0 and the molecular weight determined by mass spectrometry is 22307.8, which is consistent with the compound shown in the instant application;

G-36: the calculated molecular weight value thereof is 22251.0 and the molecular weight determined by mass spectrometry is 22252.9, which is consistent with the compound shown in the instant application;

G-38: the calculated molecular weight value thereof is 22306.0 and the molecular weight determined by mass spectrometry is 22307.2, which is consistent with the compound shown in the instant application;

G-40: the calculated molecular weight value thereof is 21534.2 and the molecular weight determined by mass spectrometry is 21535.6, which is consistent with the compound shown in the instant application;

G-42: the calculated molecular weight value thereof is 21448.1 and the molecular weight determined by mass spectrometry is 21449.3, which is consistent with the compound shown in the instant application;

G-49: the calculated molecular weight value thereof is 21447.1 and the molecular weight determined by mass spectrometry is 21448.4, which is consistent with the compound shown in the instant application.

With the above-mentioned method, fusion proteins were prepared on the basis of the insulin receptor binding polypeptide and the interleukin-1 receptor antagonistic protein, and their molecular weights were determined with mass spectrometry as follows:

IN-28: the calculated molecular weight value thereof is 24264.4 and the molecular weight determined by mass spectrometry is 24266.1, which is consistent with the compound shown in the instant application.

Preparation of 16-(1-tert-butoxy-5-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butylamino)-1,5-dioxopentyl-2-ylamino)-16-oxohexadecanoic acid

Mono-Boc-butanediamine (N-t-butoxycarbonyl-1,4-butanediamine) (1.88 g, 10 mM) and maleic anhydride (1.22 g, 12.5 mM) were dissolved in anhydrous CH₂Cl₂(30 ml), stirred at room temperature for 2 h, and filtered to collect white precipitates. The white precipitates were rinsed with CH₂Cl₂ and then desiccated under vacuum to be used directly in the next reaction.(Z)-4-(4-(tert-butoxycarbonylamino)butylamino)-4-oxobut-2-butenoic acid (2.4 g, 8.4 mM), acetic anhydride (20 ml) and anhydrous sodium acetate (1 g, 12.2 mM) were stirred at 140° C. for 6 hr. The mixture was poured into ice water and extracted with chloroform. The organic layer was washed with water and the saturated saline, dried over anhydrous magnesium sulphate, concentrated under a reduced pressure, and purified by a silica gel column (n-hexane:ethyl acetate=100/0:50/50 v/v), yielding white solid, butyl tert-butyl-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) carbamate(1.71 g, 6.4 mM).¹H NMR (300 MHz, CDCl3): δ=6.68 (s, 2H, CH═CH), 4.50 (br, 1H, NHtBoc), 3.50 (t, 2H, CH₂N), 3.08 (q, 2H, CH₂NHtBoc), 1.60-1.25 (m, 1 3H, CH₂).

Butyl tert-butyl-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) carbamate was dissolved in 4NHCl/dioxane, stirred at room temperature for 1 h, and the solvent was evaporated under a reduced pressure to yield 1-(4-aminobutyl)-1H-pyrrolyl-2,5-dione. 1-(4-aminobutyl)-1H-pyrrolyl-2,5-dione (1 g, 6 mM) was dissolved in 100 mM Na₂CO₃ (1 mL, pH 8) and acetonitrile (2 mL) at room temperature. Tert-butyl hexadecandioyl-L-Glu (OSu)-OtBu (4.09 g, 7.2 mM) was dissolved in acetonitrile (4 mL), stirred for 30 mM, acidified with 50% acetic acid, and loaded onto a RP-HPLC C5 column for purification, yielding

16-(1-tert-butoxy-5-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butylamino)-1 ,5-dioxopentyl-2-ylamino)-16-oxohexadecanoic acid.

Acylation of the protein/polypeptide (3)

The fusion protein and the equimolar amount of 16-(1-tert-butoxy-5-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl-1-yl)butylamino)-1,5-dioxopentyl-2-ylamino group)-16-oxohexadecanoic acid were dissolved in PBS with a polypeptide concentration of 10 mM. The reaction was run at room temperature for 1 h and subjected to HPLC purification. Buffer A: 0.1% TFA aqueous solution, 10% acetonitrile; buffer B: 0.1% TFA aqueous solution, 80% acetonitrile. The polypeptide initially purified and lyophilized was placed into TFA/TIS/H₂O (95:2.5:2.5, 10 mL). Thirty minutes later, the solvent was evaporated under vacuum and the crude product was dissolved in Buffer A and lyophilized. Purification was performed using the RP-HPLC C5 column, with buffer A: 0.1% TFA aqueous solution, 10% acetonitrile and buffer B: 0.1% TFA aqueous solution, 80% acetonitrile.

With the above-mentioned method, fusion proteins were prepared on the basis of the GLP-1 receptor binding polypeptide and the interleukin-1 receptor antagonistic protein, and their molecular weights were determined with mass spectrometry as follows:

G-9: the calculated molecular weight value thereof is 22177.0 and the molecular weight determined by mass spectrometry is 22179.5, which is consistent with the compound shown in the instant application;

G-12: the calculated molecular weight value thereof is 22465.2 and the molecular weight determined by mass spectrometry is 22467.1, which is consistent with the compound shown in the instant application;

G-14: the calculated molecular weight value thereof is 21258.0 and the molecular weight determined by mass spectrometry is 21259.6, which is consistent with the compound shown in the instant application;

G-15: the calculated molecular weight value thereof is 22135.9 and the molecular weight determined by mass spectrometry is 22137.3, which is consistent with the compound shown in the instant application;

G-17: the calculated molecular weight value thereof is 22423.1 and the molecular weight determined by mass spectrometry is 22424.7, which is consistent with the compound shown in the instant application;

G-25: the calculated molecular weight value thereof is 21319.0 and the molecular weight determined by mass spectrometry is 21320.4, which is consistent with the compound shown in the instant application;

G-27: the calculated molecular weight value thereof is 21634.3 and the molecular weight determined by mass spectrometry is 21636.0, which is consistent with the compound shown in the instant application;

G-29: the calculated molecular weight value thereof is 22439.2 and the molecular weight determined by mass spectrometry is 22441.1, which is consistent with the compound shown in the instant application.

With the above-mentioned method, fusion proteins were prepared on the basis of the insulin receptor binding polypeptide and the interleukin-1 receptor antagonistic protein, and their molecular weights were determined with mass spectrometry as follows:

IN-25: the calculated molecular weight value thereof is 25096.3 and the molecular weight determined by mass spectrometry is 25097.5, which is consistent with the compound shown in the instant application.

Preparation of I—CH₂—CONH—(CH₂CH₂O)₂—(CH₂)₂—NH—(N^(α)-(tBuOOC(CH₂)₁₄CO)-γ-Glu-OtBu)

Ethyl Tert-butyl-N-[2-[2-(2-aminoethoxy)ethoxy]ethylicarbamate (2.48 g, 10 mM), 2-iodoacetic acid (2.05 g, 1 1 mM), EDC (1.72 g, 11 mM), and HOBT (1.49 g, 11 mM) were dissolved in anhydrous DMF and reacted at room temperature overnight. The reaction product was washed successively with 10% of hydrogen chloride aqueous solution, water, the saturated sodium hydrogen carbonate, water, and the saturated saline, dried, concentrated under a reduced pressure, and purified with a silica gel column, yielding tert-butyl-N-[2-[2-[2-[(2-iodoacetyl)amino]ethoxy] ethoxy]ethyl]carbamate. Tert-butyl-N-[2-[2-[2-[(2-iodoacetyl)amino]ethoxy] ethoxy]ethyl]carbamate was dissolved in 4NHCl/dioxane, stirred at room temperature for 1 h, and the solvent was evaporated under a reduced pressureto yield N-[2-[2-(2-aminoethoxy)ethoxy] ethyl]-2-iodo-ethylamide hydrochloride which can be used directly in the next reaction. N-[2-[2-(2-aminoethoxy)ethoxy] ethyl]-2-iodo-ethylamide hydrochloride (1 g, 2.8 mM) was dissolved in 100 mM Na₂CO₃ (1 mL, pH 8) and acetonitrile (2 mL) at room temperature. Tert-butyl hexadecandioyl-L-Glu (OSu)-OtBu (1.42 g, 2.5 mM) was dissolved in acetonitrile (4 mL), stirred for 30 min, acidified with 50% acetic acid, and loaded onto a RP-HPLC C5 column for purification, yielding the final product, tert-butyl-16-[[1-(tert-butoxy)carbonyl-4-[2-[2-[2-[(2-iodo acetyl) amino]ethoxy]ethoxy] ethylaminol-4-oxo-butyl]amino]-16-oxo-palmitate. The calculated molecular weight is 825.4, and the determined molecular weight: 826.9.

I—CH₂—CONH—(CH₂CH₂O)₄—(CH₂)₂—NH—(N^(α)-(tBuOOC(CH₂)₁₆CO)-γ-Glu-OtBu) was prepared by a similar procedure.

Acylation of the protein/polypeptide (4)

The fusion protein and the equimolar amount of I—CH₂—CONH—(CH₂CH₂O)₂—(CH₂)₂—NH—(N^(α)-(HOOC(CH₂)₁₄CO)-γ-Glu-OtBu) were dissolved in 50 mM TrisHCl, 5 mM EDTA, pH 8.0 and reacted at room temperature in the dark for 90 minutes. Purification by HPLC. Buffer A: 0.1% TFA aqueous solution, 10% acetonitrile; buffer B: 0.1% TFA aqueous solution, 80% acetonitrile. The polypeptide initially purified and lyophilized was placed into TFA/TIS/H₂O (95:2.5:2.5, 10 mL). Thirty minutes later, the solvent was evaporated under vacuum and the crude product was dissolved in Buffer A and lyophilized Purification was performed using the RP-HPLC CS column, with buffer A: 0.1% TFA aqueous solution, 10% acetonitrile and buffer B: 0.1% TFA aqueous solution, 80% acetonitrile.

With the above-mentioned method, fusion proteins were prepared on the basis of the GLP-1 receptor binding polypeptide and the interleukin-1 receptor antagonistic protein, and their molecular weights were determined with mass spectrometry as follows:

G-32: the calculated molecular weight value thereof is 22512.3 and the molecular weight determined by mass spectrometry is 22513.4, which is consistent with the compound shown in the instant application;

G-33: the calculated molecular weight value thereof is 22471.3 and the molecular weight determined by mass spectrometry is 22473.1, which is consistent with the compound shown in the instant application;

G-34: the calculated molecular weight value thereof is 22443.2 and the molecular weight determined by mass spectrometry is 22445.0, which is consistent with the compound shown in the instant application;

G-35: the calculated molecular weight value thereof is 22512.3 and the molecular weight determined by mass spectrometry is 22513.2, which is consistent with the compound shown in the instant application;

G-37: the calculated molecular weight value thereof is 22485.3 and the molecular weight determined by mass spectrometry is 22487.1, which is consistent with the compound shown in the instant application; G-39: the calculated molecular weight value thereof is 22628.3 and the molecular weight determined by mass spectrometry is 22630.6, which is consistent with the compound shown in the instant application;

G-41: the calculated molecular weight value thereof is 21768.5 and the molecular weight determined by mass spectrometry is 21770.4, which is consistent with the compound shown in the instant application;

G-43: the calculated molecular weight value thereof is 21640.3 and the molecular weight determined by mass spectrometry is 21642.7, which is consistent with the compound shown in the instant application;

G-44: the calculated molecular weight value thereof is 21582.3 and the molecular weight determined by mass spectrometry is 21583.9, which is consistent with the compound shown in the instant application;

G-45: the calculated molecular weight value thereof is 21798.5 and the molecular weight determined by mass spectrometry is 21800.4, which is consistent with the compound shown in the instant application;

G-46: the calculated molecular weight value thereof is 21611.3 and the molecular weight determined by mass spectrometry is 21612.9, which is consistent with the compound shown in the instant application;

G-47: the calculated molecular weight value thereof is 21640.3 and the molecular weight determined by mass spectrometry is 21642.2, which is consistent with the compound shown in the instant application;

G-48: the calculated molecular weight value thereof is 21785.5 and the molecular weight determined by mass spectrometry is 21787.6, which is consistent with the compound shown in the instant application;

G-50: the calculated molecular weight value thereof is 21681.4 and the molecular weight determined by mass spectrometry is 21682.8, which is consistent with the compound shown in the instant application.

With the above-mentioned method, fusion proteins were prepared on the basis of the insulin receptor binding polypeptide and the interleukin-1 receptor antagonistic protein, and their molecular weights were determined with mass spectrometry as follows:

IN-26: the calculated molecular weight value thereof is 25089.4 and the molecular weight determined by mass spectrometry is 25091.6, which is consistent with the compound shown in the instant application.

Modification of the Polypeptide by Polyethylene Glycol (PEGylation):

1. the amino group (at the N-terminus of the main chain or on the side chain of a lysine)

a) Reductive Alkylation

The fusion protein, mPEG20K-CHO, sodium cyanoborohydride (NaBH₃CN) in a ratio of 1:2:45 were dissolved in a solution of acetic acid, pH 4.3 (0.1 M NaCl, 0.2 M CH₃COOH, 0.1 M Na₂CO₃). Protein concentration is 0.5-1 mg/mL. The reaction was monitored and purified by HPLC. Yield is about 55%. Polyethylene glycol can be selectively attached at the N-terminus of the polypeptide through reductive alkylation.

b) Acylation of NHS Ester (N-Hydroxyl Succinimide)

The fusion protein and mPEG20K-NHS in a molar ratio of 1:1 was dissolved in a solution of 0.1 NN,N-Bis(2-hydroxyethyl) glycine (pH 8), with the protein concentration of 0.5 mg/mL. The reaction was run at room temperature for 2 hours and subjected to HPLC purification. Yield is approximately 90%.

2. Mercapto Group (Cysteine)

a) PEG-maleimide

The fusion protein and the equimolar amount of mPEG20K-maleimide were dissolved in PBS (NaCl 150 mM, phosphate 20 mM, pH 7.5) with a protein concentration of 3 mg/ml. The reaction was run at room temperature for 1 h and subjected to HPLC purification. Yield is approximately 90%.

b) PEG-iodoethylamide

The fusion protein (3 mg/ml) and PEG-iodoethylamide (1.5 equivalent) were dissolved in 50 mM Tris HCl, 5 mM EDTA, pH 8.0 and reacted at room temperature in the dark for 90 minutes. The unreacted PEG reagent was removed by a D-Salt™ Dextran desalting column and the resultant was purified by HPLC.

With the above-mentioned method, fusion proteins were prepared on the basis of the GLP-1 receptor binding polypeptide-the interleukin-1 receptor antagonistic protein-PEG, and their molecular weights were determined with mass spectrometry as follows:

G-10: the calculated molecular weight value thereof is 41584.2 and a wide peak with an intermediate molecular weight of 41590.4 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

G-11: the calculated molecular weight value thereof is 41091.7 and a wide peak with an intermediate molecular weight of 41097.1 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

G-13: the calculated molecular weight value thereof is 40719.3 and a wide peak with an intermediate molecular weight of 40713.6 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

G-16: the calculated molecular weight value thereof is 41926.5 and a wide peak with an intermediate molecular weight of 41931.3 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

G-23: the calculated molecular weight value thereof is 40522.1 and a wide peak with an intermediate molecular weight of 40530.2 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

G-24: the calculated molecular weight value thereof is 40780.3 and a wide peak with an intermediate molecular weight of 40786.1 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

G-26: the calculated molecular weight value thereof is 41095.6 and a wide peak with an intermediate molecular weight of 41100.5 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

G-28: the calculated molecular weight value thereof is 41410.9 and a wide peak with an intermediate molecular weight of 41413.4 is determined by mass spectrometry, which is consistent with the compound shown in the instant application.

With the above-mentioned method, fusion proteins were prepared on the basis of the insulin receptor binding polypeptide- the interleukin-1 receptor antagonistic protein-PEG, and their molecular weights were determined with mass spectrometry as follows:

IN-27: the calculated molecular weight value thereof is 43939.9 and a wide peak with an intermediate molecular weight of 43947.8 is determined by mass spectrometry, which is consistent with the compound shown in the instant application.

G-52 synthesis

The IL-1ra and the equimolar amount of maleimide-PEG11-maleimide were dissolved in PBS with the polypeptide concentration of 5 mM. The reaction was run at room temperature for 1 h, and then the equimolar amount of HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGA PPPC (SEQ ID NO:185) was added. Two hours later, the reaction product was purified by HPLC with buffer A: 0.1% TFA aqueous solution, 10% acetonitrile and buffer B: 0.1% TFA aqueous solution, 80% acetonitrile. The calculated molecular weight value of the compound is 22176.9 and the molecular weight determined by mass spectrometry is 22186.7, with the sequencing result consistent with the sequence shown in the instant application.

With the above-mentioned method, fusion proteins were prepared on the basis of the GLP-1 receptor binding polypeptide-PEG-the interleukin-1 receptor antagonistic protein, and their molecular weights were determined with mass spectrometry as follows:

G-51: the calculated molecular weight value thereof is 22149.9 and the molecular weight determined by mass spectrometry is 22151.4, which is consistent with the compound shown in the instant application;

G-53: the calculated molecular weight value thereof is 21406.1 and the molecular weight determined by mass spectrometry is 21408.3, which is consistent with the compound shown in the instant application;

G-54: the calculated molecular weight value thereof is 21433.1 and the molecular weight determined by mass spectrometry is 21434.6, which is consistent with the compound shown in the instant application;

G-55: the calculated molecular weight value thereof is 21335.0 and the molecular weight determined by mass spectrometry is 21335.8, which is the compound shown in the instant application;

G-56: the calculated molecular weight value thereof is 21362.0 and the molecular weight determined by mass spectrometry is 21364.5, which is consistent with the compound shown in the instant application;

G-57: the calculated molecular weight value thereof is 22108.8 and the molecular weight determined by mass spectrometry is 22110.2, which is consistent with the compound shown in the instant application;

G-58: the calculated molecular weight value thereof is 22135.8 and the molecular weight determined by mass spectrometry is 22137.0, which is consistent with the compound shown in the instant application;

G-59: the calculated molecular weight value thereof is 41303.0 and a wide peak with an intermediate molecular weight of 41324.5 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

G-60: the calculated molecular weight value thereof is 41330.0 and a wide peak with an intermediate molecular weight of 41337.9 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

G-61: the calculated molecular weight value thereof is 40515.2 and a wide peak with an intermediate molecular weight of 40522.4 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

G-62: the calculated molecular weight value thereof is 40586.2 and a wide peak with an intermediate molecular weight of 40581.3 is determined by mass spectrometry, which is the compound shown in the instant application;

G-63: the calculated molecular weight value thereof is 40488.1 and a wide peak with an intermediate molecular weight of 40478.2 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

G-64: the calculated molecular weight value thereof is 40471.0 and a wide peak with an intermediate molecular weight of 40485.6 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

G-65: the calculated molecular weight value thereof is 41261.9 and a wide peak with an intermediate molecular weight of 41273.4 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

G-66: the calculated molecular weight value thereof is 41288.9 and a wide peak with an intermediate molecular weight of 41295.7 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

G-67: the calculated molecular weight value thereof is 41417.1 and a wide peak with an intermediate molecular weight of 41426.8 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

G-70: the calculated molecular weight value thereof is 40629.3 and a wide peak with an intermediate molecular weight of 40640.4 is determined by mass spectrometry, which is the compound shown in the instant application;

G-71: the calculated molecular weight value thereof is 21434.1 and the molecular weight determined by mass spectrometry is 21435.7, which is consistent with the compound shown in the instant application;

G-72: the calculated molecular weight value thereof is 21461.1 and the molecular weight determined by mass spectrometry is 21463.0, which is consistent with the compound shown in the instant application;

G-73: the calculated molecular weight value thereof is 21362.1 and the molecular weight determined by mass spectrometry is 21363.2, which is consistent with the compound shown in the instant application;

G-74: the calculated molecular weight value thereof is 21389.1 and the molecular weight determined by mass spectrometry is 21391.5, which is consistent with the compound shown in the instant application;

G-75: the calculated molecular weight value thereof is 21290.9 and the molecular weight determined by mass spectrometry is 21292.3, which is consistent with the compound shown in the instant application;

G-76: the calculated molecular weight value thereof is 21317.9 and the molecular weight determined by mass spectrometry is 21319.1, which is consistent with the compound shown in the instant application;

G-77: the calculated molecular weight value thereof is 21290.9 and the molecular weight determined by mass spectrometry is 21292.6, which is consistent with the compound shown in the instant application;

G-78: the calculated molecular weight value thereof is 21317.9 and the molecular weight determined by mass spectrometry is 21319.5, which is consistent with the compound shown in the instant application;

G-79: the calculated molecular weight value thereof is 21773.7 and the molecular weight determined by mass spectrometry is 21775.2, which is consistent with the compound shown in the instant application;

G-80: the calculated molecular weight value thereof is 21800.7 and the molecular weight determined by mass spectrometry is 21801.8, which is consistent with the compound shown in the instant application.

Synthesis of G-68

IL-1ra was dissolved in sodium phosphate to 1 mg/ml and the pH was adjusted to 5.0.2-fold equivalents of mPEG20K-CHO (whose structure is CH₃O—(CH₂CH₂O)_(n)—(CH₂)₂—CHO) was added, 45-fold equivalents of sodium cyanoborohydride was added to the concentration of 1 mM. The mixture was allowed to react at 4° C. for 12 hrs and purified by HPLC. The PEG20K IL-1ra and the equimolar amount of maleimide-PEG11-maleimide were dissolved in PBS with the polypeptide concentration of 5 mM. The reaction was run at room temperature for 1 h, and then the equimolar amount of HGEGTFTSDL SKQMEE EAVRLFIEWLKNGGPSSGAPPPC (SEQ ID NO:185) was added. Two hours later, the reaction product was purified by HPLC with buffer A: 0.1% TFA aqueous solution, 10% acetonitrile and buffer B: 0.1% TFA aqueous solution, 80% acetonitrile. The calculated molecular weight value of the compound is 42176.9 and a wide peak with an intermediate molecular weight of 42193.4 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

G-69 was prepared in the same procedure.

G-69: the calculated molecular weight value thereof is 41406.1 and a wide peak with an intermediate molecular weight of 41424.5 is determined by mass spectrometry, which is consistent with the compound shown in the instant application.

With the above-mentioned method, fusion proteins were prepared on the basis of the GIP receptor binding polypeptide-PEG-the interleukin-1 receptor antagonistic protein, and their molecular weights were determined with mass spectrometry as follows:

GI-24: the calculated molecular weight value thereof is 23078.0 and the molecular weight determined by mass spectrometry is 23082.4, which is consistent with the compound shown in the instant application;

GI-25: the calculated molecular weight value thereof is 62255.1 and a wide peak with an intermediate molecular weight of 62283.5 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

GI-26: the calculated molecular weight value thereof is 62181.1 and a wide peak with an intermediate molecular weight of 62217.0 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

GI-27: the calculated molecular weight value thereof is 21712.5 and the molecular weight determined by mass spectrometry is 21714.6, which is consistent with the compound shown in the instant application;

GI-28: the calculated molecular weight value thereof is 23206.2 and the molecular weight determined by mass spectrometry is 23210.8, which is consistent with the compound shown in the instant application; GI-29: the calculated molecular weight value thereof is 61598.4 and a wide peak with an intermediate molecular weight of 61567.3 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

GI-30: the calculated molecular weight value thereof is 21086.8 and the molecular weight determined by mass spectrometry is 21089.1, which is consistent with the compound shown in the instant application.

Synthesis of the double chain insulin receptor binding polypeptide

The document method (Han, etal.,“Insulin chemical synthesis using a two-step orthogonal formation of the three disulfides”, 21st American Peptide Society Symposium, 2009).The A chain and the B chain were synthesized by Fmoc or Boc chemistry synthesis. The cysteines at positions A7 and B7 were protected by a general protective group, while the mercapto groups on the side chains of the cysteines at positions A6, A11, A20, and B19 were protected by Acm. After being cleaved from the resin, the synthesized A and B chains were converted into A-(SH)7(S-Acm)^(6, 11, 20) and B-(SH)⁷(S-Acm) ¹⁹.The B chain was dissolved in DMF or DMSO, and an equimolar amount of 2,2′-dithio-bis(5-nitropyridine) was added. The reaction was monitored and purified by HPLC, yielding B-(S-Npys)⁷(S-Acm)¹⁹. The equimolar amount of A-(SH)⁷(S-Acm)^(6,11,20) and B-(S-Npys)⁷(S-Acm)¹⁹ were dissolved in DMSO with a polypeptide concentration of 15 mg /mL. When an A7-B7 disulfide bond was formed, 80% acetic acid aqueous solution was added and the polypeptide concentration was diluted to 1 mg/ml. Then, 40-fold amount of I₂ was added. The reaction was stirred at room temperature for 1 hour and then the ascorbic acid aqueous solution was added to stop the reaction. The mixture was purified by HPLC, and the final product was verified by mass spectrometry.

Synthesis of IN-29

FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPRTGKGS S S AAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQ LENYC_([6])N (SEQ ID NO:283) ([1] to [6] indicates the No. of the cysteines; 3 pairs of disulfide bonds are formed by six cysteines in the compound, the specific positions for the three pairs of disulfide bonds are: C_([1]) and C_([4]) forming a disulfide bond, C_([2]) and C_([6]) forming a disulfide bond, C_([3]) and C_([5]) forming a disulfide bond) (68 mg) was dissolved in NH₄HCO₃ (15 mL, pH 9), Mal-dPEG12-NHS(10 mg) was added and stirred at room temperature for 30 min. Then, IL-1Ra (175 mg) was added and the reaction was further stirred at room temperature for 1 h.The sample was purified by HPLC, with buffer A: 0.1% TFA aqueous solution, 10% acetonitrile and buffer B: 0.1% TFA aqueous solution, 80% acetonitrile. The calculated molecular weight value of the compound is 24737.9 and the molecular weight determined by mass spectrometry is 24739.6. The sequencing result is consistent with the sequence shown in the instant application.

Synthesis of Al, B29-diBoc-insulin

Human insulin (100 mg) was dissolved in water (1 mL), NaHCO₃ (0.3 mL), and DMF (3 mL). t-Boc-azide (6 mg) was added. The mixture was stirred at 40° C. for 3 hours and 50% acetic acid (0.35 mL) was added to stop the reaction. The unreacted t-Boc-azide was extracted with ethyl ether (2×15 mL). The aqueous layer was freeze-dried under vacuum. The crude product comprised the insulin mono-, bi-, and tri-substituted with Boc. The mixture was purified with a SP-Sephadex C-25 ion exchange column. The ion exchange column was firstly equilibrated with 1.5 M acetic acid containing 6 M urea, the elution rate for the polypeptide is a 48 mL/h, with a linear gradient of 0.04-0.4 M sodium chloride/1000 mL of 1.5 M acetic acid containing 6 M urea. Di-t-Boc insulin was purified further with a DEAE-Sephadex A-25 column. The chromatographic column was equilibrated previously with 0.01 M Tris buffer (pH 8.5) containing 7 M urea. The elution rate was 35 mL/h, with a gradient of 0.14-0.28 M sodium chloride/100 mL of Tris buffer. The calculated molecular weight value thereof is 6007.9 and the molecular weight determined by mass spectrometry is 6009.2. A small amount of the polypeptide was dissolved in 0.05M NH₄HCO₃/20%ACN and reduced with DTT for 10 mM, following mass spectrometric analysis. A(G1-N^(α)-Boc): the calculated molecular weight value thereof is 2483.9 and the molecular weight determined by mass spectrometry is 2485.1.B(K29-N-^(ε)-Boc): the calculated molecular weight value thereof is 3530.1 and the molecular weight determined by mass spectrometry is 3532.5. After trypsin digestion, the calculated molecular weight value of the fragment not comprising Boc is 2487.9 and the molecular weight thereof determined by mass spectrometry is 2488.7; while the calculated molecular weight value of the fragment comprising Boc is 1060.2 and the molecular weight thereof determined by mass spectrometry is 1061.3. The amino group at the B1 in the A1,B29-di-Boc polypeptide can be attached to polyethylene glycol, an albumin, a fatty acid, etc. to form a long-lasting polypeptide.

Synthesis of IN-35

A1,B29-diBoc-insulin (60 mg) was dissolved in DMF (3 mL), then Mal-dPEG12-NHS(9 mg) and triethylamine (30 μL) were added. The reaction was stirred for 2 hours at room temperature. After the solvent was evaporated under a reduced pressure, the crude product was dissolved in H₂O/CAN(3:1) and purified by RP-HPLC. The calculated molecular weight value thereof is 6758.8 and the molecular weight determined by mass spectrometry is 6760.4. The maleimide-diBoc-insulin was dissolved in purified water, with a polypeptide concentration of 10 mM. IL-1Ra (172 mg) was added and incubated at 37° C. for 30 min. Then, the resultant solution was diluted with a solution of 20 mM sodium phosphate containing 5 mM sodium octanoate and 750 mM ammonium sulphate. The unreacted reagents were removed by gel filtration chromatography, using 0.05 M ammonium bicarbonate aqueous solution as the elution solution. The pure product was obtained after freeze-dry in vacuum. The calculated molecular weight value of the obtained compound is 23884.2 and the molecular weight determined by mass spectrometry is 23886.7. The compound was assayed to be Di-Boc IN-35.Di-Boc IN-35 was dissolved in TFA/TIS(95:5) (3 mL) and stirred at room temperature for 15 min, the solvent was removed under a reduced pressure, and the crude product was purified by RP-HPLC with buffer A: 0.1% TFA aqueous solution, 10% acetonitrile and buffer B: 0.1% TFA aqueous solution, 80% acetonitrile. The calculated molecular weight value of the final compound is 23684.9 and the molecular weight determined by mass spectrometry is 23887.5, and was assayed to be IN-35.

With the above-mentioned method, fusion proteins were prepared on the basis of the insulin receptor binding polypeptide -the interleukin-1 receptor antagonistic protein -PEG, and their molecular weights were determined with mass spectrometry as follows:

IN-30: the calculated molecular weight value thereof is 24710.9 and the molecular weight determined by mass spectrometry is 24712.3, which is consistent with the compound shown in the instant application;

IN-31: the calculated molecular weight value thereof is 43987.1 and a wide peak with an intermediate molecular weight of 43993.5 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

IN-32: the calculated molecular weight value thereof is 43960.1 and a wide peak with an intermediate molecular weight of 43972.6 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

IN-35: the calculated molecular weight value thereof is 23684.9 and the molecular weight determined by mass spectrometry is 23685.2, which is consistent with the compound shown in the instant application;

IN-36: the calculated molecular weight value thereof is 23981.3 and the molecular weight determined by mass spectrometry is 23983.4, which is consistent with the compound shown in the instant application;

IN-37: the calculated molecular weight value thereof is 42934.1 and a wide peak with an intermediate molecular weight of 42945.8 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

IN-38: the calculated molecular weight value thereof is 23657.9 and the molecular weight determined by mass spectrometry is 23659.0, which is consistent with the compound shown in the instant application;

IN-39: the calculated molecular weight value thereof is 42907.1 and a wide peak with an intermediate molecular weight of 42915.6 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

IN-40: the calculated molecular weight value thereof is 23954.3 and the molecular weight determined by mass spectrometry is 23957.1, which is consistent with the compound shown in the instant application;

IN-41: the calculated molecular weight value thereof is 23583.8 and the molecular weight determined by mass spectrometry is 23585.6, which is consistent with the compound shown in the instant application;

IN-42: the calculated molecular weight value thereof is 42833.0 and a wide peak with an intermediate molecular weight of 42834.4 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

IN-43: the calculated molecular weight value thereof is 23556.8 and the molecular weight determined by mass spectrometry is 23559.2, which is consistent with the compound shown in the instant application;

IN-44: the calculated molecular weight value thereof is 42806.0 and a wide peak with an intermediate molecular weight of 42812.5 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

IN-45: the calculated molecular weight value thereof is 24825.0 and the molecular weight determined by mass spectrometry is 24826.8, which is consistent with the compound shown in the instant application;

IN-46: the calculated molecular weight value thereof is 44074.2 and a wide peak with an intermediate molecular weight of 44083.1 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

IN-47: the calculated molecular weight value thereof is 24798.0 and the molecular weight determined by mass spectrometry is 24799.3, which is consistent with the compound shown in the instant application;

IN-48: the calculated molecular weight value thereof is 44047.2 and a wide peak with an intermediate molecular weight of 44056.9 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

IN-49: the calculated molecular weight value thereof is 24783.9 and the molecular weight determined by mass spectrometry is 24785.7, which is consistent with the compound shown in the instant application;

IN-50: the calculated molecular weight value thereof is 44033.1 and a wide peak with an intermediate molecular weight of 44026.3 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

IN-51: the calculated molecular weight value thereof is 24825.0 and the molecular weight determined by mass spectrometry is 24827.5, which is consistent with the compound shown in the instant application;

IN-52: the calculated molecular weight value thereof is 44074.2 and a wide peak with an intermediate molecular weight of 44081.6 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

IN-53: the calculated molecular weight value thereof is 24841.0 and the molecular weight determined by mass spectrometry is 24843.1, which is consistent with the compound shown in the instant application;

IN-54: the calculated molecular weight value thereof is 44090.2 and a wide peak with an intermediate molecular weight of 44098.0 is determined by mass spectrometry, which is consistent with the compound shown in the instant application;

IN-55: the calculated molecular weight value thereof is 24135.3 and the molecular weight determined by mass spectrometry is 24139.2, which is consistent with the compound shown in the instant application;

IN-56: the calculated molecular weight value thereof is 24108.3 and the molecular weight determined by mass spectrometry is 24117.5, which is consistent with the compound shown in the instant application;

IN-57: the calculated molecular weight value thereof is 23772.0 and the molecular weight determined by mass spectrometry is 23773.9, which is consistent with the compound shown in the instant application;

IN-58: the calculated molecular weight value thereof is 24068.4 and the molecular weight determined by mass spectrometry is 24070.7, which is consistent with the compound shown in the instant application;

IN-59: the calculated molecular weight value thereof is 23670.9 and the molecular weight determined by mass spectrometry is 23672.8, which is consistent with the compound shown in the instant application;

IN-60: the calculated molecular weight value thereof is 23903.2 and the molecular weight determined by mass spectrometry is 23891.3, which is consistent with the compound shown in the instant application;

IN-61: the calculated molecular weight value thereof is 23802.1, and the molecular weight determined by mass spectrum is 23812.6, which is consistent with the compound shown in the instant application.

Receptor Competitive Binding Assay

1. Receptor binding assay for IL-1Ra

Reference was made to “Interleukin-1 receptor antagonist activity of a human interleukin-1 inhibitor”, Hannum et al, Nature 343, 336-340). Briefly, a standard amount of ³⁵S-labeled IL-1Ra with a specific activity of 4000 Ci/mmol was added to a 96-well plate to reach a final concentration which approximately equals to its K_(d) (150 pM). Murine EL4 thymoma cells (ATCC, TIB181, about 5000 receptors per cell) or Chinese hamster ovary (CHO)derived cells expressing human IL-1 receptor (about 30,000 receptors per cell) and the fusion protein of different concentrations (diluted in series from 20 mM to 20 pM) were cultured at 4° C. for 4 hours. The cells were harvested through a Millipore millititer plate filter system, and radioactivity retained on the filter membrane was counted by an Ambis radioanalytical imaging system. Percentage of wild type activity was defined as IC₅₀ (wild type)/IC₅₀ (fusion protein). The K_(d) for wild type IL-1ra was estimated using a simplification mode of the Cheng-Prusoff relationship (K_(d)=IC₅₀/2) and ranged from 150 to 400 pM, consistent with the values previously reported in publications.

2. Receptor Binding Assay for Insulin

(1) Preparation of ¹²⁵I-insulin

According to the document method (Cresto, et al., “Preparation of biologically active mono-¹²⁵I-insulin of high specific activity.” Acta Physiol Lat Am. 1981,31(1):13-24)

(2) Receptor binding Assay for the Compound

According to the document method (E. K. Frandsen and R. A. Bacchus. “New, simple insulin-receptor assay with universal application to solubilized insulin receptors and receptors in broken and intact cells.” Diabetes,1987,36,3:335-340) or one of the following methods. Unless otherwise stated, the method of preparing receptor was the same as the literature method, using human placenta membrane. Typically, 0.025 mg of placenta membrane was used in insulin receptor binding assay.

In the insulin receptor binding assay, the starting standard concentrations of both the insulin and the fusion proteins of the present invention were 100 nM, and the insulin and the fusion proteins were 3-fold diluted serially, resulting in the solutions with seven different concentrations (100 nM, 33.33 nM, 11.11 nM, 3.70 nM, 1.23 nM, 0.41 nM, 0.13 nM, and 0.04 nM). For the fusion proteins having an activity for the insulin receptor lower than 10% of human insulin standard, the starting concentration of the proteins was 500 nM.

Truncated Water-Soluble Receptors

Insulin receptor, ¹²⁵I-insulin (3 pM) and a series of fusion proteins diluted 3-fold were added toa buffer [100 mM Hepes, pH 8.0, 100 mM NaCl, 10 mM MgCl₂, 0.5% (w/v) BSA, 0.025% (w/v) Triton X-100] to reach a total volume of 200 μL, incubated for 48 h at 4° C. Subsequently, the receptor and fusion proteins and ligand bound to the receptor were precipitated with 0.2% γ-globulin and 500 μL of 25% (w/v) PEG 8000, and the radioactivity in the precipitate was determined. The concentration of the receptor was adjusted to a concentration at which 15-20% of receptor binds to the ligand when no fusion proteins was added.

Membrane-Bound Receptors

Membrane-bound receptors used in receptor binding assays were from BHK cells overexpressing full-length insulin receptors. Equal number of transfected BHK cells (2000-5000) was evenly seeded in each well of a 96-well plate and grown for 24 h in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal bovine serum before performing the receptor binding assay. Cells were firstly washed once with binding buffer (DMEM containing 0.50% BSA, 20 mM Hepes, pH 7.8), then¹²⁵I-insulin (6.5 pM) and a series of fusion proteins diluted 3-foldin binding buffer were added. Cultured for 3 h at 16° C. and unbound polypeptides were sucked out using a sucker, and washed once with 1.2 ml of binding buffer. Cells were solubilized in 500 μl of 1% (w/v) SDS, 100 mM NaCl, 25 mM Hepes (pH 7.8) and detected. The number of cells was adjusted to a number of which 16-28% of receptor binds to the ligand when no fusion proteins was added.

Insulin Receptor: ¹²⁵I-Insulin (30 nCi), a series of fusion proteins diluted 3-fold and placental membranes (0.025 mg) were incubated in 0.05 ml of buffer described above for 1 h at 20° C. Samples were filtered with EHWP filters, and the incubation tubes and filters were washed four times with 2.5 ml of cold buffer free of bovine albumin. Less than 5% of the fusion proteins were attached to the filters in the absence of placental membranes. Nonspecific binding of ligand to placental membranes was measured by adding excess amount of non-iodonation insulin (1 μM) to the incubation mixture. Nonspecific binding usually accounts for less of 1% of the total binding of ligand to placental membranes.

Specific binding percentage=(binding radioactivity-nonspecific binding radioactivity/total binding radioactivity-nonspecific binding radioactivity)×100. Total binding radioactivity is the total radioactivity detected before addition of fusion proteins and binding radioactivity is the radioactivity detected after addition of fusion proteins. IC₅₀ of fusion proteins was calculated using Origin software (OriginLab, Northampton, Mass.). Activity of fusion protein relative to human insulin standard=IC₅₀ human insulin standard/IC₅₀ fusion protein.

3. GLP-1cAMP Assay

BHK (baby hamster kidney) cells expressing the GLP-1 receptor of human pancreatic islet were prepared according to the literature(Knudsen and Pridal, 1996, Eur. J. Pharm. 318, 429-435). The plasma membrane was prepared according to the literature (Adelhorst, et al., 1994, J. Biol. Chem. 269, 6275), homogenized in a buffer (10 mmol/1 Tris-HCl, 30 mmol/1 NaCl, pH 7.4, 1 mmol DTT, 5 mg/l leupeptin, 5 mg/l pepstatin, 100 mg/l bacitracin, 16 mg/l aprotinin). The homogeneous mixture was centrifuged over a layer of 41 w/v % sucrose. The white zone between the upper and lower layers was dissolved in a buffer and centrifuged. The plasma membrane was stored at −80° C.

A 96-well microtiter plate was used in the analysis assay, with a total volume of 140 μl. The buffer contained 50 mmol/1 Tris-HCl, pH 7.4, 1 mmol/l EGTA, 1.5 mmol/l MgSO4, 1.7 mmol/l ATP, 20 mM GTP, 2 mmol/, 3-isobutyl-1-methylxanthine, 0.01% Tween-20, 0.1% human serum albumin The tested sample was dissolved and diluted with the buffer, and added to the membrane preparation. The mixture was incubated at 37° C. for 2 hours. The reaction was stopped by adding 25 μl of 0.05 mol/l HCl. After 10-fold diluted, the samples was measured with scintillation proximity assay (SPA) for cAMP. For references, see Kahl, et al.,“Scintillation Proximity Assay.” February, 2005. DOI: 10.1002/0471142301.ns0715s30. http://www.currentprotocols.com/WileyCDA/CPUnit/refld-ns0715.html.

EC₅₀ for the GLP-1 (7-37) standard was 61 pM.

4. GIP cAMP Assay

The assay was done as described in the publication (Wheeler et al , “Characterization of the carboxyl-terminal domain of the rat glucose-dependent insulinotropic polypeptide (GIP) receptor: a role for serines 426 and 427 in internalization”J. Biol. Chem. 1999, 274: 24593-24601). CHO cells stably expressing GIP receptors were transferred to a 96-well microtiter plate, incubated for 48 hours. The cells were washed with a 37° C. analytic buffer buffered by HEPES (DMEM/F12, 15 mM HEPES (Sigma-Aldrich), 0.1% bovine albumin (Sigma-Aldrich), pH 7.4), pre-incubated for 60 minutes. The samples (0.001˜100 nM fusion protein dissolved in 10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 25 mM NaHCO₃, 0.5 mM3-isobutyl-1-methylxanthine, and 1%(w/v) bovine albumin) were added to the cells, and cultured for 30 minutes. For GIP receptor antagonists, cells were first incubated with the antagonists for 15 min, then 1 nM GIP(1-42) was added and cultured for 30 minutes. The amount of cAMP was measured with scintillation proximity assay.

5. GIP Receptor Binding Assay

GIP (5 μg) was iodinated by the traditional chloramine-T method and purified with C-18 column (Sep-Pak; Millipore Corp.), the gradient of acetonitrile was 30-45%. Specific activity of radioactively labeled GIP ranged from 10 to 50 μCi/mg. ¹²⁵I-GIP was dissolved in analytical buffer with a concentration of 3×10⁵cpm/100 μl. CHO cells expressing GIP receptors (1-5×10⁵/well) were washed twice with the binding buffer (DMEM/F12, 20 mm HEPES, 0.1% bovine albumin, 0.5 mg/ml bacitracin, pH 7.4). ¹²⁵I-GIP (50000 cpm) and 0.3-500 nM fusion protein were added, and incubated for 12-16 h at 4° C. ¹²⁵I-GIP unbound to the receptor was separated in vacuum. The microtiter plate were washed with ice cold PBS containing 0.1% (w/v) bovine albumin and dried at room temperature. 30 μl Ultima Gold(Perkin-Elmer) could be added according to the actual conditions, ¹²⁵I-GIP content was measured by a gamma-ray counter. IC₅₀ calculation method is the same as that in the insulin receptor binding assay. IC₅₀ of GIP (1-42) standard was 2.1±0.75 nM.

Experimental Results:

The biological activity of the compounds of the present invention was detected by performing GLP-1 receptor binding capability, insulin receptor binding capability, GIP receptor binding capability, and interleukin-1 receptor binding capability assays on these compounds, using the human GLP-1 (7-37) as the reference for the GLP-1 receptor binding capacity (100%), the human insulin as the reference for the insulin receptor binding capacity (100%), the GIP (1-42) as the reference for the GIP receptor binding capacity (100%), and the wild-type human IL-1Ra as the reference for the interleukin-1 receptor antagonistic protein (100%). The obtained results were shown in Tables 1, 2, and 3, respectively.

TABLE 1 The biological activity of the fusion protein of the GLP-1 receptor binding polypeptide and the interleukin-1 receptor antagonistic protein GLP-1 Il-1ra receptor receptor No. (%) (%) G-1 52 33 G-2 53 40 G-3 61 46 G-4 67 45 G-5 45 37 G-6 49 48 G-7 50 51 G-8 48 53 G-9 30 26 G-10 29 30 G-11 21 33 G-12 22 34 G-13 19 31 G-14 20 32 G-15 17 26 G-16 25 33 G-17 20 32 G-18 42 31 G-19 43 38 G-20 55 46 G-21 58 45 G-22 60 47 G-23 27 24 G-24 32 27 G-25 33 29 G-26 26 30 G-27 24 32 G-28 23 35 G-29 43 34 G-30 57 42 G-31 53 40 G-32 62 41 G-33 54 43 G-34 46 42 G-35 53 40 G-36 60 44 G-37 63 46 G-38 61 43 G-39 62 42 G-40 51 45 G-41 46 41 G-42 45 42 G-43 41 40 G-44 40 40 G-45 46 42 G-46 45 43 G-47 42 41 G-48 42 40 G-49 43 42 G-50 44 43 G-51 92 47 G-52 90 46 G-53 79 46 G-54 78 47 G-55 72 45 G-56 70 44 G-57 83 45 G-58 82 43 G-59 81 39 G-60 80 37 G-61 75 38 G-62 73 36 G-63 71 37 G-64 70 34 G-65 77 36 G-66 76 35 G-67 52 35 G-68 51 33 G-69 45 34 G-70 42 32 G-71 78 46 G-72 77 47 G-73 78 46 G-74 76 45 G-75 71 46 G-76 70 43 G-77 82 44 G-78 81 42 G-79 120 40 G-80 115 39 G-81 39 12 G-82 7 12 G-83 37 8 G-84 15 9

TABLE 2 The biological activity of the fusion protein of the insulin receptor binding polypeptide and the interleukin-1 receptor antagonistic protein Insulin receptor IL-1 No. (%) receptor IN-1 17 35 IN-2 17 34 IN-3 16 35 IN-4 20 39 IN-5 20 38 IN-6 18 37 IN-7 25 49 IN-8 25 47 IN-9 24 47 IN-10 26 50 IN-11 25 48 IN-12 25 48 IN-13 13 34 IN-14 12 32 IN-15 12 32 IN-16 18 42 IN-17 18 40 IN-18 17 40 IN-19 23 47 IN-20 23 44 IN-21 21 43 IN-22 25 46 IN-23 25 46 IN-24 24 45 IN-25 19 38 IN-26 18 39 IN-27 10 31 IN-28 9 29 IN-29 53 45 IN-30 53 42 IN-31 9 33 IN-32 9 31 IN-33 16 40 IN-34 15 44 IN-35 19 45 IN-36 10 34 IN-37 14 30 IN-38 50 47 IN-39 14 33 IN-40 12 35 IN-41 47 46 IN-42 13 31 IN-43 45 43 IN-44 11 32 IN-45 20 39 IN-46 10 28 IN-47 19 38 IN-48 9 26 IN-49 27 25 IN-50 16 20 IN-51 49 37 IN-52 8 19 IN-53 47 36 IN-54 9 18 IN-55 38 35 IN-56 38 36 IN-57 20 23 IN-58 7 17 IN-59 21 19 IN-60 21 24 IN-61 23 22 IN-62 18 47 IN-63 17 45 IN-64 20 42 IN-65 17 43 IN-66 15 41 IN-67 11 46 IN-68 5 10 IN-69 7 18 IN-70 8 42

TABLE 3 The biological activity of the fusion protein of the GIP receptor binding polypeptide and the interleukin-1 receptor antagonistic protein GIP receptor IL-1 receptor No. (%) (%) GI-1 53 46 GI-2 264  47 GI-3 298  44 GI-4 312  45 GI-5 316  42 GI-6 (50) 48 GI-7 (53) 45 GI-8 (55) 43 GI-9 (55) 40 GI-10   (6.2) 42 GI-11 187  38 GI-12 179  41 GI-13 356  39 GI-14 368  40 GI-15 381  45 GI-16 (57) 43 GI-17 (61) 47 GI-18 (72) 46 GI-19 (54) 50 GI-20 (65) 48 GI-21 (63) 45 GI-22 36 21 GI-23 (28) 19 GI-24 137  50 GI-25 24 18 GI-26 (19) 23 GI-27 42 67 GI-28 79 51 GI-29 156  24 GI-30 (49) 58

Note: The cAMP assay was used for analysis of GI-1 to GI-5, GI-11 to GI-15, GI-22, GI-24, GI-25, and GI-27 to GI-29 in the tables. The GIP receptor binding assay was used for analysis of other proteins, wherein the ratio of the activity of the proteins vs. GIP (1-42) in the GIP receptor binding assay was shown in the parenthesis. These proteins are inhibitors to the GIP receptor. Accordingly, the receptor binding assay was used instead of the cAMP assay.

Animal Test

1. The 5 week-old C57BL/6J mice was fed high-fat/high-sucrose feedstuff(in which 58% of calories came from fat, 26% from carbohydrate, and 16% from protein). The mice were assigned into four groups, six animals per group. The mice in Group 1 were intraperitoneally injected daily with the physiological saline (0.9%w/v, NaCl), the mice in Group 2 were injected daily with IL-1Ra (500 nmol/kg), and the mice in Groups 3 and 4 were injected daily with the fusion proteins G-2 and G-20 (50 nmol/kg), for 12 consecutive weeks, followed by a glucose tolerance test. The mice were first fasted for 12 hours and then intraperitoneally injected with glucose (2 mg/g). Blood was collected at 0, 15, 30, 60, 90, 120 min respectively and the blood glucose was measured. Furthermore, the levels of serum insulin were measured at 0 and 30 min respectively.

In the test shown in FIG. 7, better blood glucose control was demonstrated in the IL-Ra group as compared with that in the physiological saline group. Furthermore, the glucose tolerance in the mice of G-2 and G-20 groups was markedly enhanced, with a low peak of blood glucose and a rapid decrease of the blood glucose level. During 12 weeks of treatment, dosages of the G-2 and G-20 were only 1/10 of the dosage of IL-1Ra, demonstrating that the fusion protein of the GLP-1 receptor binding polypeptide and the interleukin-1 receptor antagonistic protein has an in vivo biological activity and a therapeutic effect significantly superior to those of the IL-1Ra.

In the test shown in FIG. 8, the fasting serum insulin level in the IL-Ra group was significantly lower than that in the physiological saline group. The insulin level in the physiological saline group varied minimally during the glucose tolerance test, while the insulin level in the IL-1Ra group was 2-fold higher than that of fasting period. The fasting serum insulin levels in the G-2 and G-20 groups were lowest, while the insulin levels during the glucose tolerance test were 2.5 to 4-fold higher than those of fasting period, suggested that the G-2 and G-20 have better regulation and protection on the function of pancreatic islet than IL-1Ra alone used. Better blood glucose control was demonstrated.

2. The 5 week-old C57BL/6J mice was fed high-fat/high-sucrose feedstuff(in which 58% of calories came from fat, 26% from carbohydrate, and 16% from protein). The mice were assigned into four groups, six animals per group. The mice in Group 1 (the control group) were intraperitoneally injected daily with the physiological saline (0.9%w/v, NaCl), the mice in Group 2 were injected daily with IL-1Ra (500 nmol/kg), and the mice in Groups 3 and 4 were injected daily with the fusion proteins IN-7 and IN-62 (30 nmol/kg) after a meal, for 12 consecutive weeks, followed by an insulin tolerance test. The mice were first fasted for 5 hours and then intraperitoneally injected with human insulin (0.75 U/kg). Blood was collected at 0, 15, 30, 60, 90, 120 min and the blood glucose was measured respectively.

The high-fat food often leads to hyperinsulinemia, resulting in a decreased sensitivity to the insulin, which is the etiologic cause in a substantial proportion of obese patients with type 2 diabetes. Increasing the insulin sensitivity is one of important methods to prevent or even treat diabetes. In the test shown in FIG. 9, better insulin sensitivity was demonstrated in the IL-Ra group as compared with that in the physiological saline group. The insulin sensitivity of the mice in the IN-7 and IN-62 groups was more superior. During 12 weeks of treatment, dosages of the IN-7 and IN-62 were only 1/17 of the dosage of IL-1Ra, demonstrating that the fusion protein of the insulin receptor binding polypeptide and the interleukin-1 receptor antagonistic protein has an in vivo biological activity and a therapeutic effect significantly superior to those of the IL-1Ra.

3. The 5 week-old C57BL/6J mice was fed high-fat/high-sucrose feedstuff (in which 58% of calories came from fat, 26% from carbohydrate, and 16% from protein). The mice were assigned into four groups, six animals per group. The mice in Group 1 (the control group) were intraperitoneally injected daily with the physiological saline (0.9%w/v, NaCl), the mice in Group 2 were injected daily with IL-1Ra (500 nmol/kg), and the mice in Groups 3 and 4 were injected daily with the fusion proteins GI-3 and GI-7 (50 nmol/kg), for 12 consecutive weeks, followed by the glucose tolerance test. The mice were first fasted for 12 hours and then intraperitoneally injected with glucose (2 mg/g). Blood was collected at 0, 15, 30, 60 min and the blood glucose was measured respectively.

In the test shown in FIG. 10, better blood glucose control was demonstrated in the IL-Ra group as compared with that in the control group with physiological saline. Furthermore, the glucose tolerance in the mice of GI-3 and GI-7 groups was markedly enhanced, with a low peak of blood glucose. During 12 weeks of treatment, dosages of the GI-3 and GI-7 were only 1/10 of the dosage of IL-1Ra, demonstrating that the fusion protein of the GIP receptor binding polypeptide and the interleukin-1 receptor antagonistic protein has an in vivo biological activity and a therapeutic effect significantly superior to those of the IL-1Ra.

While the fusion proteins in the above-mentioned tests were not certainly superior to the standard proteins (IL-1Ra, GLP(7-37), the human insulin, and GIP (1-42)) in the corresponding receptor-binding assays(Tables 1, 2, and 3), the fusion proteins exhibited in vivo more excellent biological activities and therapeutic effects through the synergism of the two constituent proteins/polypeptides, which verifies the correctness of methods of designing and preparing the fusion proteins in the present invention. The fusion proteins in the present invention are a promising class of novel compounds for prophylaxis and treatment of diabetes. 

1. A protein obtained by linking an interleukin-1 receptor antagonist protein or an analogue thereof with another polypeptide which is an insulin receptor binding polypeptide or an analogue thereof; the structure of said protein is as follows: the interleukin-1 receptor antagonist protein or an analogue thereof -L_(j)-another polypeptide, or another polypeptide -L_(j)-the interleukin-1 receptor antagonist protein or an analogue thereof; the sequence of said interleukin-1 receptor antagonist protein or an analogue thereof is X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPI EPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVX_(IL84)ITDLSENRKQDKRFAFIRS DSGPTTSFESAAX_(IL116)PGWFLX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE(SE Q ID NO:204), wherein X_(IL0) is absent, a cysteine, general formula 1, general formula 2, or methionine; X_(IL66), X_(IL69), X_(IL116), X_(IL122) is a cysteine or serine; X_(IL84) is a cysteine or asparagine; the structure of general formula 1 is

in which J is the structure of L_(n)-M_(L) or a hydrogen atom; M_(L) is a modifying group selected from -W-X-Y-Z, a fatty acid, polyethylene glycol, albumin, IgG Fc, glycosyl; L_(n) is a selectable linking group, covalent linkage or absent; the selectable linking group is selected from polyethylene glycol, a long chain fatty acid, a polypeptide, a natural or non-natural amino acid, or a long chain formed by linking one or more of polyethylene glycol molecules, fatty acids, amino acid molecules through covalent bonds; the structure of -W-X-Y-Z wherein W is an α-amino acid residue having a carboxyl group in the side chain, this residue forms, with one of its carboxyl groups, an amide group together with the amino group at the end of L_(n); or W is a chain composed of two, three or four α-amino acids linked together via amide bonds, this chain is linked via an amide bond to the amino group at the end of L_(n), the amino acid residues of W being selected from the group of amino acid residues having a neutral side chain and/or amino acid residues having a carboxyl group in the side chain so that W has at least one amino acid residue which has a carboxyl group in the side chain; or or W is a covalent bond from X to the amino group at the end of L_(n); X is —CO—, —CH(COOH)CO—, —N(CH₂COOH)CH₂ CO—, —N(CH₂COOH)CH₂CON—(CH₂COOH)CH₂ CO—, —N(CH₂CH₂COOH)CH₂CH₂ CO—, —NHCH(COOH)(CH₂)₄NHCO—, —N(CH₂CH₂COOH)CH₂CH₂CON(CH₂CH₂COOH)CH₂CH₂ CO—, —N(CH₂CH₂COOH)CH₂ CO— or —N—(CH₂COOH)CH₂CH₂ CO—, wherein a) the above-mentioned X forms an amide bond with an amino group in W through the bond of the underlined carbonyl C, when W is an amino acid residue or a series of amino acid residues, or b) the above-mentioned X forms an amide bond with the amino group at the end of L_(n) through the bond of the underlined carbonyl C, when W is a covalent bond; Y is —(CH₂)_(m), wherein m is an integer from 6 to 32; and Z is —COOH, —CO-Asp, —CO-Glu, —CO-Gly, —CO-Sar, —CH(COOH)₂, —N(CH₂COOH)₂, —SO₃H, —PO₃H or absent, provided that Z is not —COOH when W is a covalent bond and X is —CO—; the structure of general formula 2 is

wherein O_(L) is M_(r)-L_(r)-NH-J, M_(r)-L_(r)-Z₁, M_(r)-L_(r)-M_(L), or a hydrogen atom; J and M_(L) are defined as the above; M_(r) is a functional group capable of reacting with the mercapto group to form a covalent linkage; L_(r) is an alternative linking group, covalent linkage, or absent, the alternative linking group is selected from polyethylene glycol, a long chain fatty acid, or a long chain formed by linking together one or more polyethylene glycol molecules and long chain fatty acid molecules through covalent bonds; Z₁ is —COOH, —CO-Asp, —CO-Glu, —CO-Gly, —CO-Sar, —CH(COOH)₂, —N(CH₂COOH)₂, —SO₃H, —PO₃H or absent; L_(j) is a linking group or spacer group, including a long chain fatty acid, polyethylene glycol, an amino acid, a short peptide, a protein, or a long chain formed by linking one or more of the optional long chain fatty acids, polyethylene glycol, amino acids, and short peptides through a covalent bond, or any of the structures connecting the two proteins/polypeptides through a covalent bond, or absent; the insulin receptor binding polypeptide includes insulin analogues, insulin derivatives as well as insulin conjugates, and other polypeptides which bind insulin receptor; the insulin receptor binding polypeptide has a biological activity for an insulin receptor, amounting to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or higher of the biological activity of the human insulin, respectively.
 2. The protein of claim 1, wherein the sequence of said interleukin-1 receptor antagonist protein or an analogue thereof is (SEQ ID NO: 431) U_(L)-RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKID VVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVX_(IL84) ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFLX_(IL122)T AMEADQPVSLTNMPDEGVMVTKFYFQEDE; or (SEQ ID NO: 432) U_(L)-CRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEK IDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVNITD LSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLX_(IL122)TAMEADQPV SLTNMPDEGVMVTKFYFQEDE; (SEQ ID NO: 433) U_(L)-RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEK IDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVNITD LSENRKQDKRFAFIRSDSGPTTSFESAASPGWFLX_(IL122)TAMEADQPV SLTNMPDEGVMVTKFYFQEDEC; (SEQ ID NO: 219) CRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVV PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFA FIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQ EDE; (SEQ ID NO: 220) CRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVV PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFA FIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQ EDE; (SEQ ID NO: 221) CRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVV PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVCITDLSENRKQDKRFA FIRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQ EDE; (SEQ ID NO: 222) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVCITDLSENRKQDKRFAF IRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE; (SEQ ID NO: 223) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVCITDLSENRKQDKRFAF IRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE; (SEQ ID NO: 224) RPSGRCSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE; (SEQ ID NO: 225) RPSGRCSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE; (SEQ ID NO: 226) RPSGRKSCKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE; (SEQ ID NO: 227) RPSGRKSCKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE; (SEQ ID NO: 228) RPSGRKSSCMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE; (SEQ ID NO: 229) RPSGRKSSCMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQE DE; or (SEQ ID NO: 230) RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVP IEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAF IRSDSGPTTSFESAASPGWFLCTAMEADQPVSLTNMPDEGCMVTKFYFQE DE.

wherein U_(L) is a structure of -W-X-Y-Z, fatty acid, polyethylene glycol, albumin, IgG Fc, glycosyl, hydrogen atom, general formula 5, or absent; -W-X-Y-Z is defined as in claim 1; the structure of general formula 5 is

in which J is defined as in claim
 1. 3. The protein of claim 1, wherein the sequence of insulin receptor binding polypeptide or an analogue thereof is (SEQ ID NO: 393) U_(L)-X_(IN300)HLC_([1])GSHLVEALYLVC_([2])GERGFX_(IN301)X_(IN302) X_(IN303)X_(IN304)X_(IN305)X_(IN306)GX_(IN307)X_(IN308)X_(IN309)X_(IN310) X_(IN311)X_(IN312)X_(IN313)X_(IN314)X_(IN315)X_(IN316)X_(IN317)GIVEQ C_([3])C_([4])X_(IN318)SIC_([5])X_(IN319)LX_(IN320)X_(IN321)LX_(IN322) X_(IN323)YC_([6])X_(IN324)X_(IN325),

wherein X_(IN300) is a tetrapeptide of phenylalanine-valine-asparagine-glutamine, tripeptide of valine-asparagine-glutamine, a dipeptide of asparagine-glutamine, glutamine, a sequence wherein any one of the amino acid residues in the sequence of the tetrapeptide, tripeptide, and dipeptide is substituted with a lysine or arginine, or absent; X_(IN301) is a phenylalanine, histidine or tyrosine; X_(IN302) is a —NH₂, tyrosine, phenylalanine, or absent; X_(IN303) is a threonine, asparagine, or absent; X_(IN304) is a proline, lysine, glutamic acid, aspartic acid, or absent; X_(IN305) is an aspartic acid, glutamic acid, proline, arginine, lysine, absent or a structure of general formula 1; X_(IN306) is a threonine, a structure of general formula 1, or absent; X_(IN307) is a serine, alanine, glycine, lysine, a structure of general formula 1, or absent; X_(IN308) is a glycine, a structure of general formula 1, or absent; X_(IN309) is a lysine, glycine, serine, a structure of general formula 1, or absent;X_(IN310) is a lysine, glycine, serine, a structure of general formula 1, or absent; X_(IN311) is a lysine, glycine, serine, alanine, a structure of general formula 1, or absent; X_(IN312) is a lysine, arginine, alanine, proline, glycine, a structure of general formula 1, or absent; X_(IN313) is a glycine, alanine, arginine, lysine, glutamine, proline, a structure of general formula 1, or absent; X_(IN314) is an arginine, alanine, proline, threonine, glutamine, glycine, a structure of general formula 1, or absent; X_(IN315) is a proline, glutamine, arginine, glycine, absent, or a structure of general formula 1; X_(IN316) is a glutamine, threonine, arginine, glycine, absent, or a structure of general formula 1; X_(IN317) is a threonine, arginine, lysine, or absent; X_(IN318) is a threonine, histidine, arginine or a structure of general formula 1; X_(IN319) is a serine or a structure of general formula 1; X_(IN320) is a tyrosine or a structure of general formula 1; X_(IN321) is a glutamine or a structure of general formula 1; X_(IN322) is a glutamic acid or a structure of general formula 1; X_(IN323) is an asparagine or a structure of general formula 1; X_(IN324) is an aspartic acid, glycine, alanine, or a structure of general formula 1; X_(IN325) is a lysine, general formula 3, arginine-general formula 3, or absent; the structure of general formula 1 is defined as in claim 1; the structure of general formula 3 is

wherein J is defined as in claim 1 and R₁ is —OH or —NH₂; U_(L) is defined as in claim
 2. 4. The protein of claim 3, wherein the sequence of insulin receptor binding polypeptide or an analogue thereof is U_(L)-FVNQHLCGSHINEALYINCGERGFFX_(IN302)X_(IN303)X_(IN304)X_(IN305)X_(IN306)GX_(IN307)X_(IN308)X_(IN309)X_(IN310)X_(IN311)X_(IN312)X_(IN313)X_(IN314)X_(IN315)X_(IN316)X_(IN317)GIVEQCCTSICSLYQLENYCNX_(IN325)(SEQ ID NO:394), U_(L) is defined as in claim
 2. 5. The protein of claim 1, wherein the sequence of insulin receptor binding polypeptide or an analogue thereof is (SEQ ID NO: 414) U_(L)-X_(IN107)HLC_([1])GSX_(IN108)LVEALYLVC_([2])GEX_(IN109) GFX_(IN110)X_(IN111)X_(IN112)X_(IN113)X_(IN114)X_(IN115)-C_(L)-GIVEQ C_([3])C_([4])X_(IN127)SICSLYQLENYC_([6])X_(IN128)X_(IN129),

X_(IN107) is a tetrapeptide of phenylalanine-valine-asparagine-glutamine, a tripeptide of valine-asparagine-glutamine, a dipeptide of asparagine-glutamine, glutamine, a sequence wherein any one of the amino acid residues in the sequence of the tetrapeptide, tripeptide, and dipeptide is substituted with a lysine or arginine, or absent; X_(IN108) is a histidine, phenylalanine, arginine, or glutamine; X_(IN109) is an arginine, alanine, glutamic acid, or aspartic acid;) X_(IN110) is a phenylalanine, tyrosine, or histidine; X_(IN111) is a tyrosine, phenylalanine, or absent; X_(IN112) is a threonine, asparagine, or absent; X_(IN113) is a proline, lysine, glutamic acid, aspartic acid, or absent; X_(IN114) is a lysine, proline, arginine, glutamic acid, aspartic acid or absent; X_(IN115) is a threonine, or absent; C_(L) is a peptide sequence of 6-60 amino acids, wherein the amino acids are primarily selected from the group consisting of glycine, alanine, serine, threonine and proline; X_(IN127) is a threonine, histidine, or arginine; X_(IN128) is an alanine, glycine, or asparagine; X_(IN129) is a lysine, a dipeptide of arginine-lysine, or absent; U_(L) is defined as in claim
 2. 6. The protein of claim 5, wherein the sequence of insulin receptor binding polypeptide or an analogue thereof is U_(L)-X_(IN107)HLC_([1])GSHLVEALYLVC_([2])GERGFX_(IN110)X_(IN111)X_(IN112)X_(IN113)X_(IN114)X_(IN115)-C_(L)-G IVEQC_([3])C_([4])TSICSLYQLENYC_([6])X_(IN128)X_(IN129)(SEQ ID NO:415), U_(L) is defined as in claim
 2. 7. The protein of claim 6, wherein the sequence of insulin receptor binding polypeptide or an analogue thereof is (SEQ ID NO: 416) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)T- C_(L)-GIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 417) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)TGKGS SSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 418) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)TGSGK SSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 419) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)TGSGS KSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 420) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)TGSGS SKAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 421) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)TGSGS SSKAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 422) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)TGSG SSSAKAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 423) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)TGSG SSSAAKPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 424) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)TGSG SSSAAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NK; or (SEQ ID NO: 425) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPX_(IN114)TGSGSSS AAAPQTGIVEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 426) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAAKPQTGI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 427) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAKAPQTGI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 428) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSKAAPQTGI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 429) U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAAAPQTGI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])NK; or (SEQ ID NO: 430) FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFGSGSSSAAAPQTGI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N;

U_(L) is defined as in claim
 2. 8. The protein of claim 5, wherein C_(L) is (SEQ ID NO: 384) GX_(IN116)X_(IN117)X_(IN118)X_(IN119)X_(IN120)X_(IN121)X_(IN122)X_(IN123) X_(IN124)X_(IN125)X_(IN126),

wherein X_(IN116) is a lysine, cysteine, serine, or alanine; X_(IN117) is a glycine, lysine, or serine; X_(IN118) is a lysine or serine; X_(IN119) is a lysine or serine; X_(IN120) is a lysine, serine, or alanine; X_(IN121) is a glycine, lysine, arginine, alanine, proline, or absent; X_(IN122) is a glycine, alanine, arginine, lysine, glutamine, proline, or absent; X_(IN123) is an arginine, lysine, glycine, alanine, proline, threonine, glutamine, or absent; X_(IN124) is a proline, glutamine, lysine, glycine, arginine, or absent; X_(IiN125) is a glutamine, threonine, lysine, glycine, arginine, or absent; X_(IN126) is a threonine, arginine, lysine, or absent.
 9. The protein of claim 8, wherein C_(L) is GAGSSSAAAPQT (SEQ ID NO:385), GSGSSSAAAPQT(SEQ ID NO:386), GSGSSSAAPQT(SEQ ID NO:387), GSGSSSAPQT(SEQ ID NO:388), or GSGSSAPQT (SEQ ID NO389).
 10. The protein of claim 1, wherein the sequence of said protein is U_(L)-X_(IN107)HLC_([1])GSX_(IN108)LVEALYLVC_([2])GEX_(IN109)GFN_(IN110)X_(IN111)X_(IN112)X_(IN113)X_(IN114)X_(IN115)-C_(L)-GIVEQC_([3])C_([4])X_(IN127)SIC_([5])SLYQLENYC_([6])X_(IN128)X_(IN129)-L_(j)-X_(IL0)RPSGRKSSKMQAFR IWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVX_(IL84)ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFL X_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE(SEQ ID NO:395), [1] to [6] indicates the No. of the cysteines; 3 pairs of disulfide bonds are formed with six cysteines in the compound, and the specific positions of the three pairs of disulfide bonds are: C_([1]) and C_([4]) forming a disulfide bond, C_([2]) and C_([6]) forming a disulfide bond, C_([3]) and C_([5]) forming a disulfide bond; U_(L) is defined as in claim
 2. 11. The protein of claim 10, wherein the sequence of said protein is U_(L)-X_(IN107)HLC_([1])GSHLVEALYLVC_([2])GERGFX_(IN110)X_(IN111)X_(IN112)X_(IN113)X_(IN114)X_(IN115)-C_(L)-GIVE QC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])N-(GGGGS)_(m)-X_(L)-(GGGGS)_(n)-X_(IL0)RPSGRKSSKMQAFRI WDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVX_(IL84)ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFL X_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE(SEQ ID NO:396), in which m and n are 0, 1, 2, 3, 4, 5, or 6; X_(L) is a cysteine, lysine, absent, general formula 1 or general formula 2; general formula 1 and general formula 2 are defined as in claim 1, U_(L) is defined as in claim
 2. 12. The protein of claim 11, wherein the sequence of said protein is U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKT-C_(L)-GIVEQC_([3])C_([4])TSIC_([5])SLYQLEN YC_([6])N-(GGGGS)_(m)-X_(L)-(GGGGS)_(n)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVA GYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVNIT DLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFLX_(IL122)TAMEADQPVSLTNMPD EGVMVTKFYFQEDE (SEQ ID NO:397), in which m and n are 0, 1, 2, 3, 4, 5, or 6; X_(L) is a cysteine, lysine, absent, general formula 1 or general formula 2; general formula 1 and general formula 2 are defined as in claim 1, U_(L) is defined as in claim
 2. 13. The protein of claim 12, wherein the sequence of said protein is U_(L)-FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKT-C_(L)-GIVEQC_([3])C_([4])TSIC_([5])SLYQLEN YC_([6])N-(GGGGS)_(n)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVN LEEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVNITDLSENRKQD KRFAFIRSDSGPTTSFESAAX_(IL116)PGWFLX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFY FQEDE(SEQ ID NO:398), n is 0, 1, 2, 3, 4, 5, or 6, U_(L) is defined as in claim
 2. 14. The protein of claim 1, wherein the sequence of said protein is (SEQ ID NO: 399) U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLE EKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAV X_(IL84)ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFL X_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE-L_(j)-X_(IN107)HL C_([1])GSX_(IN108)LVEALYLVC_([2])GEX_(IN109)GFX_(IN110)X_(IN111) X_(IN112)X_(IN113)X_(IN114)X_(IN115)-C_(L)-GIVEQC_([3])C_([4])X_(IN127) SIC_([5])SLYQLENYC_([6])X_(IN128)X_(IN129),

in which [1] to [6] indicates the No. of the cysteines; 3 pairs of disulfide bonds are formed with six cysteines in the compound, and the specific positions of the three pairs of disulfide bonds are: C_([1]) and C_([4]) forming a disulfide bond, C_([2]) and C_([6]) forming a disulfide bond, C_([3]) and C_([5]) forming a disulfide bond; U_(L) is defined as in claim
 2. 15. The protein of claim 14, wherein the sequence of said protein is (SEQ ID NO: 400) U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLE EKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAV X_(IL84)ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFL X_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE-(GGGGS)_(m)-X_(L)- (GGGGS)_(n)-X_(IN107)HLC_([1])GSHLVEALYLVC_([2])G ERGFX_(IN110)X_(IN111)X_(IN112)X_(IN113)X_(IN114)X_(IN115)-C_(L)-GI VEQC_([3])C_([4])TSIC_([5])SLYQLENYC_([6])X_(IN128)X_(IN129); or (SEQ ID NO: 401) U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNL EEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVN ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFLX_(IL122)TA MEADQPVSLTNMPDEGVMVTKFYFQEDE-(GGGGS)_(m)-X_(L)-(GGGGS)_(n)- FVNQHLC_([1])GSHLVEALYLVC_([2])GERGFFYTPKT-C_(L)-GIVEQ C_([3]C) _([4])TSIC_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 402) U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLE EKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVN ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFLX_(IL122)TA MEADQPVSLTNMPDEGVMVTKFYFQEDE-(GGGGS)_(n)-FVNQHLC_([1])G SHLVEALYLVC_([2])GERGFFYTPKT-C_(L)-GIVEQC_([3])C_([4])TSI C_([5])SLYQLENYC_([6])N; or (SEQ ID NO: 403) U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLE EKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAV X_(IL84)ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)PGWFL X_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE-(GGGGS)_(m)- X_(L)-(GGGGS)_(n)-FVNQHLCGSHLVEALYLVCGERGFFYTPKTGSGSSSAA APQTGIVEQCCTSICSLYQLENYCN;

in which m and n are 0, 1, 2, 3, 4, 5, or 6; X_(L) is a cysteine, lysine, absent, general formula 1 or general formula 2;[1] to [6] indicates the No. of the cysteines; 3 pairs of disulfide bonds are formed with six cysteines in the compound, and the specific positions of the three pairs of disulfide bonds are: C_([1]) and C_([4]) forming a disulfide bond, C_([2]) and C_([6]) forming a disulfide bond, C_([3]) and C_([5]) forming a disulfide bond; general formula 1 and general formula 2 are defined as in claim 1, U_(L) is defined as in claim
 2. 16. The protein of claim 1, wherein the sequence of said protein is (SEQ ID NO: 410) U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLE EKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEAVN ITDLSENRKQDKRFAFIRSDSGPTTSFESAAC (the linking group-the insulin receptor binding polypeptide)PGWFLX_(IL122)TAMEADQPVSLTNMPDE GVMVTKFY FQEDE; or (SEQ ID NO: 411) U_(L)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVN LEEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLEA VC (the linking group-the insulin receptor binding polypeptide) ITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFL X_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE; or (SEQ ID NO: 412) (the insulin receptor binding polypeptide-the linking group)-C_(IL0)-X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLR NNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMX_(IL66)LS X_(IL69)VKSGDETRLQLEAVX_(IL84)ITDLSENRKQDKRF AHRSDSGPTTSFESAAX_(IL116)PGWFLX_(IL122)TAMEADQPVSLTNMPDE GVMVTKFYFQEDE, (SEQ ID NO: 413) U_(L)X_(IL0)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLE EKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSGDETRLQLE AVX_(IL84)ITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)P GWFLX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDEC_(IL153) (the linking group-the insulin receptor binding polypeptide), (SEQ ID NO: 434) (the insulin receptor binding polypeptide-the linking group)RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQ GPNVNLEEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKS GDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAAX_(IL116)P GWFLX_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE; or (SEQ ID NO: 435) (the insulin receptor binding polypeptide-the linking group)CRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYL QGPNVNLEEKIDVVPIEPHALFLGIHGGKMX_(IL66)LSX_(IL69)VKSG DETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAASPGWFL X_(IL122)TAMEADQPVSLTNMPDEGVMVTKFYFQEDE;

in which C_(IL0) and C_(IL153) are cysteine or absent; U_(L) is defined as claim
 2. 17. The protein of claim 1, wherein said protein is selected from the compounds IN-1 to IN-70.
 18. A protein conjugate, wherein said protein conjugate is a compound obtained through modifications including acylation, grafting, and conjugation on the basis of the protein of claim
 1. 19. A pharmaceutical composition comprising the protein of claim 1 or the protein conjugate thereof, as well as pharmaceutically acceptable carriers or additives.
 20. A method of treating diabetes, comprising administering to a patient in need the protein of claim 1, the protein conjugate thereof, or the pharmaceutical composition of thereof. 